Controlled Release Formulations for the Delivery of HIF-1 Inhibitors

ABSTRACT

Controlled release dosage formulations for the delivery of one or more HIF-1 inhibitors are provided. The controlled release formulations contain one or more HIF-1 inhibitors conjugated to or dispersed in a polymeric vehicle. The one or more HIF-1 inhibitors can be dispersed or encapsulated in a polymeric matrix. In some embodiments, the one or more HIF-1 inhibitors are covalently bound to a polymer, forming a polymer-drug conjugate. Polymeric vehicles can be formed into implants, microparticles, nanoparticles, or combinations thereof. Controlled release HIF-1 formulations provide prolonged therapeutic benefit while lowering side effects by releasing low levels of one or more HIF-1 inhibitors and/or HIF-1 inhibitor conjugates over a prolonged period of time. Controlled release dosage formulations can be used to treat or prevent a disease or disorder in a patient associated with vascularization, including cancer, obesity, and ocular diseases such as wet AMD.

CROSS REFERENCE TO RELATED APPLICATION

This application claims benefit of U.S. Provisional Application No.61/611,931 filed Mar. 16, 2012, which is herein incorporated byreference in its entirety.

SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with Government Support under AgreementsR01CA140746, P30EY001765, and U54CA151838 awarded to Justin Scot Hanesby the National Institutes of Health, and under Agreement R01EY012609awarded to Peter Anthony Campochiaro by the National Institutes ofHealth. The Government has certain rights in the invention.

FIELD OF THE INVENTION

The present invention relates to polymeric controlled releaseformulations for the delivery of an effective amount of one or moreHIF-1 inhibitors, in particular, to the eye, as well as methods of usethereof for the treatment and prevention of diseases, particularly forthe treatment or prevention of ocular diseases.

BACKGROUND OF THE INVENTION

Hypoxia Inducible Factor 1 (HIF-1) is a transcription factor thatcontrols expression of more than 60 target genes whose products arecritical to many processes, including angiogenesis. For example,vascular endothelial growth factor (VEGF), the most important knownregulator of angiogenesis, is upregulated by HIF-1. Active HIF-1 iscomposed of alpha (HIF-1α, 2α) and beta (HIF-1 (3) subunits thatdimerize and bind to consensus sequences (hypoxia responsive elements,HREs) in the regulatory regions of the target genes. In normoxia, HIF-1is hydroxylated and interacts with the von Hippel Lindau protein (pVHL),an E3 ubiquitin ligase subunit that targets HIF for degradation. In theabsence of oxygen, HIF hydroxylation is inhibited, preventing binding topVHL and leading to its intracellular accumulation. Increased levels ofintracellular HIF-1α and HIF-2α have been associated with many aberrantvascularization processes.

Because of its critical role in angiogenesis, HIF-1 represents apromising target for the treatment and prevention of diseases anddisorders associated with ocular neovascularization. Attempts to developclinically useful therapies have been plagued by difficulty inadministering and maintaining a therapeutically effective amount ofHIF-1 inhibitors for an extended period of time. In addition, many HIF-1inhibitors are cytotoxic, and exhibit significant side effects and/ortoxicity, especially when administered to the ocular tissue.

In order to treat chronic diseases of the eye, there is a need for longacting methods for delivering HIF-1 inhibitors to the eye. Formulationswhich provide extended delivery of HIF-1 will minimize the potential fortoxicity associated with the administration of many HIF-1 inhibitors.Formulations which provide extended delivery of HIF-1 will also sustainsuppression of VEGF and other stimulators of angiogenesis, maximizeefficacy, promote regression of neovascularization, and minimize thepotential for catastrophic complications including subretinalhemorrhage. In addition, reducing the need for frequent injections willdecrease the risk of endophthalmitis and decrease the burden of frequentclinic visits, a major hardship for patients and their families.

Therefore, it is an object of the invention to provide formulations ofHIF-1 inhibitors with improved stability, safety, and efficacy.

It is also an object of the invention to provide drug formulationscapable of effectively delivering therapeutic levels of one or moreHIF-1 inhibitors for an extended period of time.

It is a further object of the invention to provide improved methods oftreating or preventing diseases or disorders of the eye.

SUMMARY OF THE INVENTION

Controlled release dosage formulations for the delivery of one or moreHIF-1 inhibitors conjugated to or dispersed in a polymeric vehicle forcontrolled delivery are described herein. The polymeric matrix can beformed from non-biodegradable or biodegradable polymers; however, thepolymer matrix is preferably biodegradable. The polymeric matrix can beformed into implants (e.g., rods, disks, wafers, etc.), microparticles,nanoparticles, or combinations thereof for delivery. Uponadministration, the one or more HIF-1 inhibitors are released over anextended period of time, either upon degradation of the polymer matrix,diffusion of the one or more inhibitors out of the polymer matrix, or acombination thereof. By employing a polymer-drug conjugate, particlescan be formed with more controlled drug loading and drug releaseprofiles. In addition, the solubility of the conjugate can be controlledso as to minimize soluble drug concentration and, therefore, toxicity.

In preferred embodiments, the one or more HIF-1 inhibitors arecovalently bound to a polymer, forming a polymer-drug conjugate. Thepolymer-drug conjugates can then be formed into implants (e.g., rods,wafers, discs, etc), microparticles, nanoparticles, or combinationsthereof for delivery to the eye. By employing a polymer-drug conjugate,particles can be formed with more controlled drug loading and drugrelease profiles. In addition, the solubility of the conjugate can becontrolled by modifying the solubility of the polymer portion and/or thebranched point (“Y” in the chemical structure of the polymer, so as tominimize soluble drug concentration and, therefore, toxicity.

In certain embodiments, the polymer-drug conjugates are block copolymerscontaining one or more HIF-1 inhibitors covalently bonded to the blockcopolymer. In one embodiment, the conjugate has the formula:

(A-X)_(m)—Y—((Z)_(o)—(X)_(p)-(A)_(q))_(n)

wherein

A represents, independently for each occurrence, a HIF-1 inhibitor;

X represents, independently for each occurrence, a hydrophobic polymersegment;

Y is absent or represents a branch point;

Z represents, independently for each occurrence, a hydrophilic polymersegment;

o, p, and q are independent 0 or 1;

m represents the number of A-X branches and is an integer between oneand twenty; and

n represent the number of Z, Z—X, and Z—X-A branches and is an integerbetween zero and twenty, more preferably between one and twenty, withthe proviso that A is not doxorubicin when m and n are both equal toone.

Exemplary polymer-drug conjugates of this type are represented by thegeneral formulae shown below

(A-X_(m)YZ)_(n)

(A-X_(m)YZ—X)_(n)

(A-X_(m)Z—X-A)_(n)

wherein

A represents, independently for each occurrence, a HIF-1 inhibitor;

X represents, independently for each occurrence, a hydrophobic polymersegment;

Y is absent or represents a branch point;

Z represents, independently for each occurrence, a hydrophilic polymersegment;

m represents the number of A-X branches and is an integer between oneand twenty; and

n represent the number of Z, Z—X, and Z—X-A branches and is an integerbetween zero and twenty, more preferably between one and twenty, withthe proviso that A is not doxorubicin when m and n are both equal toone.

A is, independently for each occurrence, a HIF-1 inhibitor. In someinstances, the HIF-1 inhibitor is an anthracycline, such as doxorubicin(DXR) or daunorubicin (DNR).

The one or more hydrophobic polymer segments can be any biocompatible,hydrophobic polymer or copolymer. In some cases, the hydrophobic polymeror copolymer is biodegradable. Examples of suitable hydrophobic polymersinclude, but are not limited to, polyesters such as polylactic acid,polyglycolic acid, or polycaprolactone, polyanhydrides, such aspolysebacic anhydride, and copolymers of any of the above. In preferredembodiments, the hydrophobic polymer is a polyanhydride, such aspolysebacic anhydride or a copolymer thereof.

The degradation profile of the one or more hydrophobic polymer segmentsmay be selected to influence the release rate of the active agent invivo. For example, the hydrophobic polymer segments can be selected todegrade over a time period from seven days to 2 years, more preferablyfrom seven days to 56 weeks, more preferably from four weeks to 56weeks, most preferably from eight weeks to 28 weeks.

The one or more hydrophilic polymer segments can be any hydrophilic,biocompatible, non-toxic polymer or copolymer. In certain embodiments,the one or more hydrophilic polymer segments contain a poly(alkyleneglycol), such as polyethylene glycol (PEG). In particular embodiments,the one or more hydrophilic polymer segments are linear PEG chains.

In some embodiments, where both hydrophobic and hydrophilic polymersegments are present, the combined weight average molecular weight ofthe one or more hydrophilic polymer segments will preferably be largerthan the weight average molecular weight of the hydrophobic polymersegment. In some cases, the combined weight average molecular weight ofthe hydrophilic polymer segments is at least five times, more preferablyat least ten times, most preferably at least fifteen times, greater thanthe weight average molecular weight of the hydrophobic polymer segment.

The branch point, when present, can be an organic molecule whichcontains three or more functional groups. Preferably, the branch pointwill contain at least two different types of functional groups (e.g.,one or more alcohols and one or more carboxylic acids, or one or morehalides and one or more carboxylic acids). In such cases, the differentfunctional groups present on the branch point can be independentlyaddressed synthetically, permitting the covalent attachment of thehydrophobic and hydrophilic segments to the branch point in controlledstoichiometric ratios. In certain embodiments, the branch point ispolycarboxylic acid, such as citric acid, tartaric acid, mucic acid,gluconic acid, or 5-hydroxybenzene-1,2,3,-tricarboxylic acid.

In certain embodiments, the polymer-drug conjugate is formed from asingle hydrophobic polymer segment and two or more hydrophilic polymersegments covalently connected via a multivalent branch point. Exemplarypolymer-drug conjugates of this type are represented by the generalformula shown below

A—X—YZ)_(n)

wherein

A represents a HIF-1 inhibitor;

X represents a hydrophobic polymer segment;

Y represents a branch point;

Z represents, independently for each occurrence, a hydrophilic polymersegment; and

n is an integer between two and ten.

A is, independently for each occurrence, a HIF-1 inhibitor. In someinstances, the HIF-1 inhibitor is an anthracycline, such as doxorubicin(DXR) or daunorubicin (DNR).

In certain embodiments, the hydrophilic polymer segments contain apoly(alkylene glycol), such as polyethylene glycol (PEG), preferablylinear PEG chains. In some embodiments, the conjugates contain betweentwo and six hydrophilic polymer segments.

In preferred embodiments, the hydrophobic polymer is a polyanhydride,such as polysebacic anhydride or a copolymer thereof. In certainembodiments, the hydrophobic polymer segment ispoly(1,6-bis(p-carboxyphenoxy)hexane-co-sebacic acid) (poly(CPH-SA) orpoly(1,3-bis(p-carboxyphenoxy)propane-co-sebacic acid) (poly(CPP-SA).

In some embodiments, the branch point connects a single hydrophobicpolymer segment to three hydrophilic polyethylene glycol polymersegments. In certain cases, the polymer-drug conjugate can berepresented by Formula I

wherein

A is a HIF-1 inhibitor;

L represents, independently for each occurrence, an ether (e.g., —O—),thioether (e.g., —S—), secondary amine (e.g., —NH—), tertiary amine(e.g., —NR—), secondary amide (e.g., —NHCO—; —CONH—), tertiary amide(e.g., —NRCO—; —CONR—), secondary carbamate (e.g., —OCONH—; —NHCOO—),tertiary carbamate (e.g., —OCONR—; —NRCOO—), urea (e.g., —NHCONH—;—NRCONH—; —NHCONR—⁻, —NRCONR—), sulfinyl group (e.g., —SO—), or sulfonylgroup (e.g., —SOO—);

R is, individually for each occurrence, an alkyl, cycloalkyl,heterocycloalkyl, alkylaryl, alkenyl, alkynyl, aryl, or heteroarylgroup, optionally substituted with between one and five substituentsindividually selected from alkyl, cyclopropyl, cyclobutyl ether, amine,halogen, hydroxyl, ether, nitrile, CF₃, ester, amide, urea, carbamate,thioether, carboxylic acid, and aryl;

PEG represents a polyethylene glycol chain; and

X represents a hydrophobic polymer segment.

In certain embodiments, the branch point is a citric acid molecule, andthe hydrophilic polymer segments are polyethylene glycol. In such cases,the polymer-drug conjugate can be represented by Formula IA

wherein

A is a HIF-1 inhibitor;

D represents, independently for each occurrence, O or NH;

PEG represents a polyethylene glycol chain; and

X is represents a hydrophobic polymer segment.

X may be any biocompatible hydrophobic polymer or copolymer. Inpreferred embodiments, the hydrophobic polymer or copolymer isbiodegradable. In preferred embodiments, the hydrophobic polymer is apolyanhydride, such as polysebacic anhydride, or a copolymer thereof.

The polymer-drug conjugates can be used to form implants (e.g., rods,discs, wafers, etc.), nanoparticles, or microparticles with improvedproperties for controlled delivery of HIF-1 inhibitors.

Also provided are pharmaceutical compositions containing implants (e.g.,rods, discs, wafers, etc.), nanoparticles, microparticles, orcombinations thereof for the controlled release of one or more HIF-1inhibitors in combination with one or more pharmaceutically acceptableexcipients. The nanoparticles, microparticles, or combination thereofcan be formed from one or more polymer-drug conjugates, or blends ofpolymer-drug conjugates with one or more polymers. The implants (e.g.,rods, discs, wafers, etc.), nanoparticles, microparticles, orcombination thereof can also be formed from a polymeric matrix havingone or more HIF-1 inhibitors dispersed or encapsulated therein.

The pharmaceutical compositions can be administered to treat or preventa disease or disorder in a patient associated with vascularization,including cancer and obesity. In a preferred embodiment, thepharmaceutical compositions are administered to treat or prevent adisease or disorder in a patient associated with ocularneovascularization. Upon administration, the one or more HIF-1inhibitors are released over an extended period of time atconcentrations which are high enough to produce therapeutic benefit, butlow enough to avoid unacceptable levels of cytotoxicity, and whichprovide much longer release than inhibitor without conjugate.

This is demonstrated in the case of controlled release formulationscontaining anthracyclines. The intravitreal injection of anthracyclinessuppresses intraocular neovascularization; however, the effect isshort-lived because the anthracyclines are rapidly eliminated from theposterior segment of the eye. In addition, the anthracyclines are tootoxic for clinical use, as indicated by a severe reduction in scoptopicERG b-wave amplitudes and retinal degeneration. However, administrationof a controlled release formulation of an anthracycline (e.g.,nanoparticles formed from a polymer-drug conjugate defined by Formula I)provides enhanced and prolonged anti-angiogenic activity with no signsof toxicity. The controlled release HIF-1 formulation provides prolongedtherapeutic benefit in the absence of side effects by releasing lowlevels of an HIF-1 inhibitor over a prolonged period of time.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-B are bar graphs plotting the area of choroidalneovascularization (CNV) (in mm²) observed in the eyes of C57BL/6 mice14 days after rupture of their Bruch's membrane by laserphotocoagulation without administration of a HIF-1 inhibitor, and uponadministration of varying amounts of doxorubicin or daunorubicin. In thecase of values of CNV measured upon HIF-1 administration, the area ofCNV observed upon HIF-1 administration (open bars) is plotted next tothe area of CNV observed in untreated fellow eyes (FE). The barsrepresent the mean (±SEM) area of choroidal NV. FIG. 1A plots the areaof CNV (in mm²) observed in the eyes of C57BL/6 mice 14 days afterrupture of their Bruch's membrane by laser photocoagulation withoutadministration of a HIF-1 inhibitor (vehicle only injected in both eyesof the mouse (BE), left bar), and upon administration of 10, 1.0, and0.1 μg of daunorubicin (DNR). Eyes injected with 10 μg of DNR showed astatistically significant reduction in the area of CNV (P<0.001, n=10)compared to fellow eyes injected with the vehicle only. Eyes injectedwith 1.0 μg and 0.1 μg of DNR did not show a statistically significantreduction in the area of CNV (for 1.0 μg, P<0.082, n=10; for 0.1 μg,P<0.399, n=10) compared to fellow eyes injected with the vehicle only.FIG. 1B plots the area of CNV (in mm²) observed in the eyes of C57BL/6mice 14 days after rupture of their Bruch's membrane by laserphotocoagulation without administration of a HIF-1 inhibitor (vehicleonly injected in both eyes of the mouse (BE), left bar), and uponadministration of 10, 1.0, and 0.1 μg of doxorubicin (DXR, open bars).Eyes injected with 10 μg of DXR showed a statistically significantreduction in the area of CNV (P<0.001, n=10) compared to fellow eyesinjected with the vehicle only. Eyes injected with 1.0 μg and 0.1 μg ofDXR did not show a statistically significant reduction in the area ofCNV (for 1.0 μg, P<0.071, n=10; for 0.1 μg, P<0.322, n=10) compared tofellow eyes injected with the vehicle only. In both FIGS. 1A and 1B, themean area of CNV was similar in fellow eyes (FE) and eyes from mice inwhich both eyes were injected with vehicle only (BE), suggesting thatthere was no systemic effect from intraocular injections of the HIF-1inhibitor.

FIGS. 2A-B are bar graphs plotting the area of retinalneovascularization (RNV) (in mm²) observed in the eyes of C57BL/6 micewith oxygen-induced ischemic retinopathy five days after theadministration of a vehicle control (PBS buffer without a HIF-1inhibitor present), and upon administration of varying amounts ofdoxorubicin or daunorubicin. The bars represent the mean (±SEM) area ofR NV. FIG. 2A plots the area of RNV (in mm²) observed in the eyes ofC57BL/6 mice with oxygen-induced ischemic retinopathy five days afterthe administration of a vehicle control (PBS buffer without a HIF-1inhibitor present, left bar), and upon administration of 1.0, 0.1, and0.01 μg of daunorubicin (DNR). Eyes injected with 1.0 μg and 0.1 μg ofDNR showed a statistically significant reduction in the area of RNV (for1 μg, P<0.001, n=6; for 0.1 μg, P=0.013, n=8). Eyes injected with 0.01μg of DNR did not show a statistically significant reduction in the areaof RNV (P=0.930, n=6). FIG. 2B plots the area of RNV (in mm²) observedin the eyes of C57BL/6 mice with oxygen-induced ischemic retinopathyfive days after the administration of a vehicle control (PBS bufferwithout a HIF-1 inhibitor present, left bar), and upon administration of1.0, 0.1, and 0.01 μg of doxorubicin (DXR). Eyes injected with 1.0 μg ofDXR showed a statistically significant reduction in the area of RNV(P<0.001, n=8). Eyes injected with 0.1 μg and 0.01 μg of DXR did notshow a statistically significant reduction in the area of RNV (for 0.1μg, P=0.199, n=7; for 0.01 μg, P=0.096, n=8).

FIG. 3A is a graph demonstrating the efficacy of a controlled-releaseformulation of a HIF-1 inhibitor (specifically DXR-PSA-PEG₃nanoparticles) in treating CNV in a mouse model of CNV. FIG. 3A is a bargraph plotting the area of CNV (in mm²) observed in the eyes of C57BL/6mice 14 days after rupture of their Bruch's membrane by laserphotocoagulation without administration of a HIF-1 inhibitor (vehicleonly injected in both eyes of the mouse (BE), left bar), and uponadministration of 10, 1.0, and 0.1 μg of DXR-PSA-PEG₃ nanoparticles. Inthe case of values of CNV measured upon administration of varyingamounts of DXR-PSA-PEG₃ nanoparticles, the area of CNV observed uponnanoparticle administration is plotted next to the area of CNV observedin untreated fellow eyes (FE). The bars represent the mean (±SEM) areaof CNV. Eyes injected with 10 μg, 1.0 μg, and 0.1 μg of DXR-PSA-PEG₃nanoparticles all showed a statistically significant reduction in thearea of CNV (for 10 μg, P<0.001, n=10; for 1.0 μg, P=0.009, n=10; for0.1 μg, P=0.007, n=10) compared to fellow eyes injected with the vehicleonly.

FIG. 3B is a graph demonstrating the efficacy of a controlled-releaseformulation of a HIF-1 inhibitor (specifically DXR-PSA-PEG₃nanoparticles) in treating established CNV in a mouse model of CNV. Inthis case, the Bruch's membrane of C57BL/6 mice were ruptured by laserphotocoagulation, and CNV was allowed to grow for a period of sevendays. Subsequently, one cohort had the baseline area of CNV measured,and the remaining mice were treated by injection of 1 μg of DXR-PSA-PEG₃nanoparticles in one eye, and injection of vehicle only in the felloweye. After an additional seven days, the area of CNV was measured in theDXR-PSA-PEG₃ and vehicle-treated eyes. FIG. 3B is a bar graph plottingthe area of CNV (in mm²) observed seven days after administration of 1μg of DXR-PSA-PEG₃ nanoparticles (left bar) and 14 days after laserphotocoagulation rupture of Bruch's membrane. The area of CNV (in mm²)observed seven days after administration of 1 μg of DXR-PSA-PEG₃nanoparticles and 14 days after laser photocoagulation rupture ofBruch's membrane is compared with the area of CNV measured in felloweyes injected with vehicle only (center bar) 14 days after laserphotocoagulation rupture of Bruch's membrane and seven days aftervehicle injection and untreated eyes seven days after laserphotocoagulation rupture of Bruch's membrane (Base line; right bar). Astatistically significant decrease in the area of CNV (P<0.001, n=10)was observed, both relative to fellow eyes injected with vehicle only(center bar) and the base line CNV observed seven days after laserphotocoagulation rupture of Bruch's membrane in untreated eyes (rightbar), demonstrating that DXR-PSA-PEG₃ treatment not only significantlyreduced CNV (compare left and middle bars), but also mediated regressionof existing CNV (compare left and right bars).

FIG. 4 is a graph demonstrating the efficacy of a controlled-releaseformulation of a HIF-1 inhibitor (specifically DXR-PSA-PEG₃nanoparticles) in treating RNV in mice with oxygen-induced ischemicretinopathy. FIG. 4 is a bar graph plotting the area of RNV (in mm²)observed in the eyes of C57BL/6 mice with oxygen-induced ischemicretinopathy five days after the administration of a vehicle control (PBSbuffer without a HIF-1 inhibitor present, right bar), and uponadministration of 1 μg of DXR-PSA-PEG₃ nanoparticles (left bar). Thebars represent the mean (±SEM) area of RNV. A statistically significantdecrease in the area of RNV (P<0.001, n=8) was observed relative tofellow eyes injected with vehicle only.

FIGS. 5A-B are bar graphs demonstrating the ability ofcontrolled-release formulation of a HIF-1 inhibitor (specificallyDXR-PSA-PEG₃ nanoparticles) to suppress subretinal neovascularization(NV) in transgenic mice in which the rhodopsin promoter drivesexpression of VEGF in photoreceptors (rho/VEGF mice) for at least 35days. At postnatal day (P) 14, hemizygous rho/VEGF mice were given anintraocular injection of 10 μg of DXR-PSA-PEG₃ nanoparticles in one eyeand vehicle only (PBS buffer) in the fellow eye.

FIG. 5A is a bar graph plotting the area of NV (in mm²) per retinaobserved four weeks after intraocular injection of 10 μg of DXR-PSA-PEG₃nanoparticles (left bar). A statistically significant decrease in thearea of NV per retina (P=0.042, n=5) was observed relative to felloweyes injected with vehicle only.

FIG. 5B is a bar graph plotting the area of NV (in mm²) per retinaobserved five weeks after intraocular injection of 10 μg of DXR-PSA-PEG₃nanoparticles (left bar). A statistically significant decrease in thearea of NV per retina (P=0.007, n=5) was observed relative to felloweyes injected with vehicle only.

FIG. 6A is a graph showing the size distribution by volume ofmicroparticles as determined using a Coulter Multisizer. FIG. 6B is agraph showing the size distribution by volume of nanoparticles asdetermined using a Coulter Multisizer.

FIG. 7A is a graph showing the amount of drug conjugate released (nM)into the aqueous humor (AH) as a function of time (days) frommicroparticles and nanoparticles injected into the eyes of rabbits. FIG.7B is a bar graph that compares the amounts of released DXR drugconjugate (nM) in the aqueous humor (AH) and the vitreous ofparticle-injected rabbit eyes. The time (days) is 105 for thenanoparticle-treated animals and 115 for the microparticle-treatedanimals.

FIG. 8 is a graph showing release of doxorubicin (DXR) conjugate invitro (m) as a function of time (days) for polymer rods containing 10%DXR (), 30% DXR (▪) and 50% DXR (▴).

FIG. 9A is a graph showing the in vitro release profile of doxorubicin(DXR) conjugate from microparticles prepared from DXR-SA-PEG₃ (▪) andDXR-SA-CPH-PEG (▴). FIG. 9B is a graph showing the in vitro releaseprofile of doxorubicin (DXR) conjugate from microparticles prepared fromDXR-SA-CPH-PEG₃ (▴).

DETAILED DESCRIPTION OF THE INVENTION I. Definitions

“Active Agent,” as used herein, refers to a physiologically orpharmacologically active substance that acts locally and/or systemicallyin the body. An active agent is a substance that is administered to apatient for the treatment (e.g., therapeutic agent), prevention (e.g.,prophylactic agent), or diagnosis (e.g., diagnostic agent) of a diseaseor disorder. “Ophthalmic Drug” or “Ophthalmic Active Agent”, as usedherein, refers to an agent that is administered to a patient toalleviate, delay onset of, or prevent one or more symptoms of a diseaseor disorder of the eye, or diagnostic agent useful for imaging orotherwise assessing the eye.

“Effective amount” or “therapeutically effective amount,” as usedherein, refers to an amount of polymer-drug conjugate effective toalleviate, delay onset of, or prevent one or more symptoms, particularlyof a disease or disorder of the eye. In the case of age-related maculardegeneration, the effective amount of the polymer-drug conjugate delays,reduces, or prevents vision loss in a patient.

“Biocompatible” and “biologically compatible,” as used herein, generallyrefer to materials that are, along with any metabolites or degradationproducts thereof, generally non-toxic to the recipient, and do not causeany significant adverse effects to the recipient. Generally speaking,biocompatible materials are materials which do not elicit a significantinflammatory or immune response when administered to a patient.

“Biodegradable Polymer,” as used herein, generally refers to a polymerthat will degrade or erode by enzymatic action and/or hydrolysis underphysiologic conditions to smaller units or chemical species that arecapable of being metabolized, eliminated, or excreted by the subject.The degradation time is a function of polymer composition, morphology,such as porosity, particle dimensions, and environment.

“Hydrophilic,” as used herein, refers to the property of having affinityfor water. For example, hydrophilic polymers (or hydrophilic polymersegments) are polymers (or polymer segments) which are primarily solublein aqueous solutions and/or have a tendency to absorb water. In general,the more hydrophilic a polymer is, the more that polymer tends todissolve in, mix with, or be wetted by water.

“Hydrophobic,” as used herein, refers to the property of lackingaffinity for, or even repelling water. For example, the more hydrophobica polymer (or polymer segment), the more that polymer (or polymersegment) tends to not dissolve in, not mix with, or not be wetted bywater.

Hydrophilicity and hydrophobicity can be spoken of in relative terms,such as but not limited to a spectrum of hydrophilicity/hydrophobicitywithin a group of polymers or polymer segments. In some embodimentswherein two or more polymers are being discussed, the term “hydrophobicpolymer” can be defined based on the polymer's relative hydrophobicitywhen compared to another, more hydrophilic polymer.

“Nanoparticle,” as used herein, generally refers to a particle having adiameter, such as an average diameter, from about 10 nm up to but notincluding about 1 micron, preferably from 100 nm to about 1 micron. Theparticles can have any shape. Nanoparticles having a spherical shape aregenerally referred to as “nanospheres”.

“Microparticle,” as used herein, generally refers to a particle having adiameter, such as an average diameter, from about 1 micron to about 100microns, preferably from about 1 micron to about 50 microns, morepreferably from about 1 to about 30 microns. The microparticles can haveany shape. Microparticles having a spherical shape are generallyreferred to as “microspheres”.

“Molecular weight,” as used herein, generally refers to the relativeaverage chain length of the bulk polymer, unless otherwise specified. Inpractice, molecular weight can be estimated or characterized usingvarious methods including gel permeation chromatography (GPC) orcapillary viscometry. GPC molecular weights are reported as theweight-average molecular weight (Mw) as opposed to the number-averagemolecular weight (Mn). Capillary viscometry provides estimates ofmolecular weight as the inherent viscosity determined from a dilutepolymer solution using a particular set of concentration, temperature,and solvent conditions.

“Mean particle size,” as used herein, generally refers to thestatistical mean particle size (diameter) of the particles in apopulation of particles. The diameter of an essentially sphericalparticle may refer to the physical or hydrodynamic diameter. Thediameter of a non-spherical particle may refer preferentially to thehydrodynamic diameter. As used herein, the diameter of a non-sphericalparticle may refer to the largest linear distance between two points onthe surface of the particle. Mean particle size can be measured usingmethods known in the art, such as dynamic light scattering.

“Monodisperse” and “homogeneous size distribution” are usedinterchangeably herein and describe a population of nanoparticles ormicroparticles where all of the particles are the same or nearly thesame size. As used herein, a monodisperse distribution refers toparticle distributions in which 90% or more of the distribution lieswithin 15% of the median particle size, more preferably within 10% ofthe median particle size, most preferably within 5% of the medianparticle size.

“Pharmaceutically Acceptable,” as used herein, refers to compounds,carriers, excipients, compositions, and/or dosage forms which are,within the scope of sound medical judgment, suitable for use in contactwith the tissues of human beings and animals without excessive toxicity,irritation, allergic response, or other problem or complication,commensurate with a reasonable benefit/risk ratio.

“Branch point,” as used herein, refers to a portion of a polymer-drugconjugate that serves to connect multiple hydrophilic polymer segmentsto one end of the hydrophobic polymer segment or multiple hydrophobicpolymer segments to one end of the hydrophilic segment.

“HIF-1 inhibitor,” as used herein, refers to, a drug that reduces thelevel of HIF-1 and/or its ability to stimulate the transcription ofgenes that contain a hypoxia response element in their promoter region.

“Implant,” as generally used herein, refers to a polymeric device orelement that is structured, sized, or otherwise configured to beimplanted, preferably by injection or surgical implantation, in aspecific region of the body so as to provide therapeutic benefit byreleasing one or more HIF-1 inhibitors over an extended period of timeat the site of implantation. For example, intraocular implants arepolymeric devices or elements that are structured, sized, or otherwiseconfigured to be placed in the eye, preferably by injection or surgicalimplantation, and to treat one or more diseases or disorders of the eyeby releasing one or more HIF-1 inhibitors over an extended period.Intraocular implants are generally biocompatible with physiologicalconditions of an eye and do not cause adverse side effects. Generally,intraocular implants may be placed in an eye without disrupting visionof the eye.

Ranges of values defined herein include all values within the range aswell as all sub-ranges within the range. For example, if the range isdefined as an integer from 0 to 10, the range encompasses all integerswithin the range and any and all subranges within the range, e.g., 1-10,1-6, 2-8, 3-7, 3-9, etc.

II. Polymer-Drug Conjugates

Controlled release conjugates including one or more HIF-1 inhibitorsconjugated to or dispersed in a polymeric vehicle for controlled releaseof the one or more HIF-1 inhibitors are provided. By administeringcontrolled release conjugates of HIF-1 inhibitors, activity is enhancedand prolonged while toxicity is reduced or eliminated.

In some embodiments, one or more HIF-1 inhibitors are dispersed orencapsulated in a polymeric matrix for delivery to the eye. Thepolymeric matrix can be formed from non-biodegradable or biodegradablepolymers;

however, the polymer matrix is preferably biodegradable. The polymericmatrix can be formed into implants, microparticles, nanoparticles, orcombinations thereof for delivery to the eye. Upon administration, theone or more HIF-1 inhibitors are released over an extended period oftime, either upon degradation of the polymer matrix, diffusion of theone or more inhibitors out of the polymer matrix, or a combinationthereof. By employing a polymer-drug conjugate, particles can be formedwith more controlled drug loading and drug release profiles.

In some embodiments, the controlled-release formulation containsparticles formed from one or more polymer-drug conjugates. Thepolymer-drug conjugates are block copolymers containing one or moreHIF-1 inhibitors covalently bonded to the block copolymer. Typically,the polymer-drug conjugates contain a HIF-1 inhibitor, one or morehydrophobic polymer segments, and one or more hydrophilic polymersegments. In certain cases, one or more hydrophilic polymer segments areattached to the one or more hydrophobic polymer segments by a branchpoint. By employing a polymer-drug conjugate, particles can be formedwith more controlled drug loading and drug release profiles. Inaddition, the solubility of the conjugate can be controlled so as tominimize soluble drug concentration and, therefore, toxicity.

In certain embodiments, the polymer-drug conjugate contains one or moreHIF-1 inhibitors covalently attached to a bioerodible polymeric segment.Preferably, the bioerodible segment to which the HIF-1 inhibitor isattached is composed of one or more monomers that possess low solubilityin aqueous solution. In certain embodiments, one or more of the monomerspossesses a solubility of less than 2 g/L, more preferably less than 1g/L, more preferably less than 0.5 g/L, most preferably less than 0.3g/L in water.

A. Structure of the Polymer-Drug Conjugates

Polymer-drug conjugates are provided which contain a HIF-1 inhibitorcovalently attached to a block copolymer.

In one embodiment, the conjugate has the formula:

(A-X)_(m)—Y—((Z)_(o)—(X)_(p)-(A)_(q))_(n)

wherein

A represents, independently for each occurrence, a HIF-1 inhibitor;

X represents, independently for each occurrence, a hydrophobic polymersegment;

Y is absent or represents a branch point;

Z represents, independently for each occurrence, a hydrophilic polymersegment;

o, p, and q are independent 0 or 1;

m represents the number of A-X branches and is an integer between oneand twenty; and

n represent the number of Z, Z—X, and Z—X-A branches and is an integerbetween zero and twenty, more preferably between one and twenty, withthe proviso that A is not doxorubicin when m and n are both equal toone.

Exemplary polymer-drug conjugates are represented by the generalformulae shown below:

(A-X_(m)YZ)_(n)

(A-X_(m)YZ—X)_(n)

(A-X_(m)YZ—X-A)_(n)

wherein

A represents, independently for each occurrence, a HIF-1 inhibitor;

X represents, independently for each occurrence, a hydrophobic polymersegment;

Y is absent, or represents a branch point;

Z represents, independently for each occurrence, a hydrophilic polymersegment; and

m represents the number of A-X branches and is an integer between oneand twenty; and

n represents the number of Z, Z—X, and Z—X-A branches and is an integerbetween zero and twenty, more preferably between one and 20, with theproviso that A is not doxorubicin when m and n are both equal to one.

A is, independently for each occurrence, a HIF-1 inhibitor. In someinstances, the active agent is an anthracycline, such as doxorubicin(DXR) or daunorubicin (DNR).

The one or more hydrophobic polymer segments can be any biocompatible,hydrophobic polymer or copolymer. In some cases, the hydrophobic polymeror copolymer is biodegradable. Examples of suitable hydrophobic polymersinclude, but are not limited to, polyesters such as polylactic acid,polyglycolic acid, or polycaprolactone, polyanhydrides, such aspolysebacic anhydride, and copolymers thereof. In preferred embodiments,the hydrophobic polymer is a polyanhydride, such as polysebacicanhydride or a copolymer thereof.

The degradation profile of the one or more hydrophobic polymer segmentsmay be selected to influence the release rate of the active agent invivo. For example, the hydrophobic polymer segments can be selected todegrade over a time period from seven days to 2 years, more preferablyfrom seven days to 56 weeks, more preferably from four weeks to 56weeks, most preferably from eight weeks to 28 weeks.

The one or more hydrophilic polymer segments can be any hydrophilic,biocompatible, non-toxic polymer or copolymer. In certain embodiments,the one or more hydrophilic polymer segments contain a poly(alkyleneglycol), such as polyethylene glycol (PEG). In particular embodiments,the one or more hydrophilic polymer segments are linear PEG chains.

In some cases, the polymer-drug conjugate contains only one hydrophilicpolymer segment (i.e., n is equal to one). In preferred embodiments, thepolymer-drug conjugate contains more than one hydrophilic polymer chain(i.e., n is greater than one). In certain embodiments, the polymer-drugconjugate contains between two and six, more preferably between threeand five hydrophilic polymer chains. In one embodiment, the polymer drugconjugate contains three hydrophilic polymer segments.

In cases where both hydrophobic and hydrophilic polymer segments arepresent, the combined molecular weight of the one or more hydrophilicpolymer segments will preferably be larger than the molecular weight ofthe hydrophobic polymer segment. In some cases, the combined molecularweight of the hydrophilic polymer segments is at least five times, morepreferably at least ten times, most preferably at least fifteen times,greater than the molecular weight of the hydrophobic polymer segment.

The branch point, when present, can be an organic molecule whichcontains three or more functional groups. Preferably, the branch pointwill contain at least two different types of functional groups (e.g.,one or more alcohols and one or more carboxylic acids, or one or morehalides and one or more carboxylic acids or one or more amines)). Insuch cases, the different functional groups present on the branch pointcan be independently addressed synthetically, permitting the covalentattachment of the hydrophobic and hydrophilic segments to the branchpoint in controlled stoichiometric ratios. In certain embodiments, thebranch point is polycarboxylic acid, such as citric acid, tartaric acid,mucic acid, gluconic acid, or 5-hydroxybenzene-1,2,3,-tricarboxylicacid.

In certain embodiments, the polymer-drug conjugate is formed from asingle hydrophobic polymer segment and two or more hydrophilic polymersegments covalently connected via a multivalent branch point. Exemplarypolymer-drug conjugates of this type are represented by the generalformula shown below

A-X—YZ)_(n)

wherein

A represents, independently for each occurrence, a HIF-1 inhibitor;

X represents, a hydrophobic polymer segment;

Y represents a branch point;

Z represents, independently for each occurrence, a hydrophilic polymersegment; and

n is an integer between zero and 300, more preferably between zero andfifty, more preferably between zero and thirty, most preferably betweenzero and ten.

A is, independently for each occurrence, a HIF-1 inhibitor. In someinstances, the HIF-1 inhibitor is an anthracycline, such as doxorubicin(DXR) or daunorubicin (DNR).

The hydrophobic polymer segment can be any biocompatible hydrophobicpolymer or copolymer. In some cases, the hydrophobic polymer segment isalso biodegradable. Examples of suitable hydrophobic polymers include,but are not limited to, copolymers of lactic acid and glycolic acid,polyanhydrides, poylcaprolactone, and copolymers thereof. In certainembodiments, the hydrophobic polymer is a polyanhydride, such aspolysebacic anhydride.

The one or more hydrophilic polymer segments can be any hydrophilic,biocompatible, non-toxic polymer or copolymer. The hydrophilic polymersegment can be, for example, a poly(alkylene glycol), a polysaccharide,poly(vinyl alcohol), polypyrrolidone, or copolymers thereof. Inpreferred embodiments, the one or more hydrophilic polymer segments are,or are composed of, polyethylene glycol (PEG).

In certain embodiments, the polymer-drug conjugate contains between twoand six, more preferably between three and five, hydrophilic polymerchains. In one embodiment, the polymer drug conjugate contains threehydrophilic polymer segments.

The branch point can be, for example, an organic molecule which containsmultiple functional groups. Preferably, the branch point will contain atleast two different types of functional groups (e.g., an alcohol andmultiple carboxylic acids, or a carboxylic acid and multiple alcohols).In certain embodiments, the branch point is polycarboxylic acid, such asa citric acid molecule.

In some embodiments, the branch point connects a single hydrophobicpolymer segment to three hydrophilic polyethylene glycol polymersegments. In certain cases, the polymer-drug conjugate can berepresented by Formula I

wherein

A is a HIF-1 inhibitor;

L represents, independently for each occurrence, an ether (e.g., —O—),thioether (e.g., —S—), secondary amine (e.g., —NH—), tertiary amine(e.g., —NR—), secondary amide (e.g., —NHCO—; —CONH—), tertiary amide(e.g., —NRCO—; —CONR—), secondary carbamate (e.g., —OCONH—; —NHCOO—),tertiary carbamate (e.g., —OCONR—; —NRCOO—), urea (e.g., —NHCONH—;—NRCONH—; —NHCONR—⁻, —NRCONR—), sulfinyl group (e.g., —SO—), or sulfonylgroup (e.g., —SOO—);

R is, individually for each occurrence, an alkyl, cycloalkyl,heterocycloalkyl, alkylaryl, alkenyl, alkynyl, aryl, or heteroarylgroup, optionally substituted with between one and five substituentsindividually selected from alkyl, cyclopropyl, cyclobutyl ether, amine,halogen, hydroxyl, ether, nitrile, CF₃, ester, amide, urea, carbamate,thioether, carboxylic acid, and aryl;

PEG represents a polyethylene glycol chain; and

X represents a hydrophobic polymer segment.

In certain embodiments, the branch point is a citric acid molecule, andthe hydrophilic polymer segments are polyethylene glycol. In such cases,the polymer-drug conjugate can be represented by Formula IA

wherein

A is a HIF-1 inhibitor;

D represents, independently for each occurrence, O or NH;

PEG represents a polyethylene glycol chain; and

X is represents a hydrophobic polymer segment.

In some embodiments, D is, in every occurrence, O. In other embodiments,D is, in every occurrence, NH. In still other embodiments, D is,independently for each occurrence, O or NH.

In some embodiments, the polymer drug conjugate is defined by thefollowing formula

A-X

wherein

A is a HIF-1 inhibitor; and

X is a hydrophobic polymer segment, preferably a polyanhydride.

B. HIF-1 Inhibitors

The polymer-drug conjugates contain one or more HIF-1 inhibitors. Anysuitable HIF-1 inhibitor may be incorporated into the polymer-drugconjugates. Preferably, the one or more HIF-1 inhibitors are HIF-1αinhibitors. The inhibitors can be small molecules and/or a biomoleculeor macromolecule (e.g., proteins, enzymes, nucleic acids, growthfactors, polysaccharides, lipids, etc.). In some instances, the smallmolecule active agent has a molecular weight of less than about 2000g/mol, preferably less than about 1500 g/mol, more preferably less thanabout 1200 g/mol, most preferably less than about 1000 g/mol. In otherembodiments, the small molecule active agent has a molecular weight lessthan about 500 g/mol. Biomolecules typically have a molecular weight ofgreater than about 2000 g/mol and may be composed of repeat units suchas amino acids (peptide, proteins, enzymes, etc.) or nitrogenous baseunits (nucleic acids).

In a preferred embodiment, the one or more HIF-1 inhibitors areanthracyclines. Anthracyclines, such as doxorubicin (DXR) anddaunorubicin (DNR), are cancer chemotherapeutic agents that bind to DNAand suppress proliferation of cancer cells. Independent of theiractivity on cell proliferation, DXR and DNR suppress transcriptionalactivity of HIF-1.

Anthracyclines are glycosides whose aglycone is a tetracyclicanthraquinone derivative. Encompassed within the term anthracycline arecompounds of the anthracycline class of natural products, as well assynthetic or semi-synthetic analogs and derivatives thereof, prodrugsthereof, and pharmaceutically acceptable salts thereof. “Analogs” and“derivatives” as used herein typically refers to compounds which retainthe anthracycline core, i.e., the tetracyclic core, but differ in one ormore functional groups attached to the core. In some embodiments, thederivatives and analogs differ in the aglycone moiety and/or the sugarresidue attached to the molecule.

Any anthracycline which provides therapeutic benefit may be incorporatedinto the polymer-drug conjugates. Preferably, the anthracycline is acompound which is used or suitable for clinical use as an antineoplasticagent in cancer chemotherapy. Examples of natural products in theanthracycline class include daunorubicin and doxorubicin, which areproduced by microorganisms belonging to the genus Streptomyces.

Examples of preferred anthracyclines include doxorubicin, daunorubicin,13-deoxydoxorubicin (also known as GPX-100), iodoxorubicin, epirubicin,THP-adriamycin, idarubicin, menogaril, aclacinomycin A (also known asaclarubicin), zorubicin, pirarubicin, valrubicin, amrubicin,iodoxorubicin, nemorubicin,(4R)-1-(4-carboxy-1-oxobutyl)-4-hydroxy-L-prolyl-L-alanyl-L-seryl(2R)-2-cyclohexylglycyl-L-glutaminyl-L-seryl-L-leucine(also known as L 377202), 4′ deoxy-4′-iododoxorubicin. Additionalanthracyclines that can be incorporated in polymer-drug conjugates areknown in the art. See, for example, Suarato, et al. Chimicaoggi, 9-19(April 1990); J W Lown: Pharmac. Ther. 60:185-214 (1993); F M Arcamone:Biochimie, 80, 201-206 (1998); C Monneret: Eur. J. Med. Chem. 36:483-493 (2001); and U.S. Pat. Nos. 4,438,015, 4,672,057, 5,646,177,5,801,257, and 6,284,737.

The polymer-drug conjugate can also contain a pharmaceuticallyacceptable salt of an anthracycline. In some cases, it may be desirableto incorporate a salt of an anthracycline into a polymer-drug conjugatedue to one or more of the salt's advantageous physical properties, suchas enhanced stability or a desirable solubility or dissolution profile.Salts of anthracycline compounds can be prepared using standard methodsknown in the art. Lists of suitable salts can be found, for example, inRemington's Pharmaceutical Sciences, 20th ed., Lippincott Williams &Wilkins, Baltimore, Md., 2000, p. 704.

The polymer-drug conjugate can also contain a prodrug of ananthracycline. Anthracycline prodrugs are compounds that can beconverted to a biologically active anthracycline, either in vivo afteradministration or in vitro prior to administration of the compound.Examples of suitable anthracycline prodrugs are known in the art. See,for example, U.S. Pat. No. 5,710,135 Leenders, et al., U.S. Pat. No.6,268,488 by Barbas, III, et al., WO 92/19639 by J. lacquesy et al., K.Bosslet et al. Cancer Res. 54: 2151-2159 (1994), S. Andrianomenjanaharyet al. Bioorg. Med. Chem. Lett. 2:1093-1096 (1992) and J.-P. Gesson etal. Anti-Cancer Drug Des. 9: 409-423 (1994).

Other suitable HIF-1 inhibitors include, without limitation, polyamidessuch as echinomycin (NSC-13502) (Kong, et al., Cancer Research. 65(19):9047-9055 (2005) and Olenyuk et al, Proc Natl Acad Sci USA 101:1676816773 (2004)), which inhibit the interaction between HIF and DNA;epidithiodiketopiperazines, such as chetomin (Kung, et al., Cancer Cell.6(3): 33-43 (2004)), which inhibit the interaction between HIF and p300;benzoazaheterocycles, such as YC-1(3-(5′-hydroxymethyl-2′-furyl)-1-benzylindazole) (Yeo, et al., Journalof the National Cancer Institute. 95(7): 516-525 (2003)); radicicol andanalogs thereof such as KF58333 (Kurebayashi, et al., Cancer Research92:1342-1351 (2001)); rapamycins, including rapamycin and analogsthereof such as, temsirolimus (CCI779) and Everolimus (RAD001) (Majumderet al. Nature Medicine 10: 594-601 (2004)); geldanamycins, includinggeldanamycin and analogs thereof such as 17-allylamino-17-demethoxygeldanamycin (17-AAG), and17-dimethylaminoethylamino-17-demethoxy-geldanamycin (17-DMAG);quinocarmycin monocitrate (KW2152) and its hydrocyanization productDX-52-1 (NSC-607097) (Rapisarda, et al. Cancer Research. 62(15):43164324 (2002)); camptothecin analogs (topoisomerase I inhibitors)(Rapisarda, et al., Cancer Research 62(15): 4316-4324 (2002)), such astopotecan (NSC609699), camptothecin, 20-ester(S) (NSC-606985), and.9-glycineamido-20(S)-camptothecin HCl (NSC-639174); microtubuledisrupting agents (Escuin, et al. Cancer Research 65(19): 9021-9028(2005)), such as paclitaxel, docetaxel, 2-methoxyestradiol, vincristine,discodermolide, and epothilone B; thioredoxin inhibitors (Welsh, et al.Molecular Cancer Therapeutics. 2:235243 (2003)), such as PX-12(1-methylpropyl 2-imidazolyl disulfide) and Pleurotin; P13-KinaseInhibitors (Jiang, et al. Cell Growth and Differentiation 12: 363-369(2001)), such as wortmannin and LY294002; protein kinase-1 (MEK-1)inhibitors such as PD98059 (2′-amino-3′ methoxyflavone); PX-12(1-methylpropyl 2-imidazolyl disulfide); pleurotin PX-478; quinoxaline1,4-dioxides; sodium butyrate (NaB); sodium nitropurruside (SNP); 103D5R(Tan, et al., Cancer Research 65: 605-612 (2005)); PX-478(S-2-amino-3-[4V-N,N,-bis(2-chloroethyl)amino]-phenyl propionic acidN-oxide dihydrochloride) (Welsh, et al., Molecular Cancer Therapeutics3: 233-244 (2004)); histone deacetylase inhibitors such as [(E)-(1S,4S,10S,21R)-7-[(Z)-ethylidene]-4,21-diisopropyl-2-oxa-12,13-dithia-5,8,20,23-tetraazabicyclo-[8,7,6]-tricos-16-ene-3,6,9,19,22-pentanone(FR901228, depsipeptide) and FK228 (FR901228) (NSC 630176) (Mie-Lee, etal., Biochemical and Biophysical Research Communications 300(1): 241-246(2003)); genistein; indanone; staurosporin; coumarins; barbituric andthiobarbituric acid analogs; (aryloxyacetylamino)benzoic acid analogs,2-methoxyestradiol and analogs thereof, digoxin and other cardiacglycosides (Zhang, et al. PNAS 105:19579), acriflavin (Lee, et al. PNAS106:17910), and hydroxamic acid compounds.

In certain embodiments, the HIF-1 inhibitor is rapamycin, temsirolimus,everolimus, geldanamycin, echinomycin, doxorubicin, daunorubicin,camptothecin, topotecan, irinotecan, or bortezomib.

C. Hydrophobic Polymer Segment

Polymer-drug conjugates can contain one or more hydrophobic polymersegments. The hydrophobic polymer segments can be homopolymers orcopolymers.

In preferred embodiments, the hydrophobic polymer segment is abiodegradable polymer. In cases where the hydrophobic polymer isbiodegradable, the polymer degradation profile may be selected toinfluence the release rate of the active agent in vivo. For example, thehydrophobic polymer segment can be selected to degrade over a timeperiod from seven days to 2 years, more preferably from seven days to 56weeks, more preferably from four weeks to 56 weeks, most preferably fromeight weeks to 28 weeks.

Examples of suitable hydrophobic polymers include polyhydroxyacids suchas poly(lactic acid), poly(glycolic acid), and poly(lacticacid-co-glycolic acids); polyhydroxyalkanoates such aspoly3-hydroxybutyrate or poly4-hydroxybutyrate; polycaprolactones;poly(orthoesters); polyanhydrides; poly(phosphazenes);poly(hydroxyalkanoates); poly(lactide-co-caprolactones); polycarbonatessuch as tyrosine polycarbonates; polyamides (including synthetic andnatural polyamides), polypeptides, and poly(amino acids);polyesteramides; polyesters; poly(dioxanones); poly(alkylene alkylates);hydrophobic polyethers; polyurethanes; polyetheresters; polyacetals;polycyanoacrylates; polyacrylates; polymethylmethacrylates;polysiloxanes; poly(oxyethylene)/poly(oxypropylene) copolymers;polyketals; polyphosphates; polyhydroxyvalerates; polyalkylene oxalates;polyalkylene succinates; poly(maleic acids), as well as copolymersthereof.

In preferred embodiments, the hydrophobic polymer segment is apolyanhydride. The polyanhydride can be an aliphatic polyanhydride, anunsaturated polyanhydride, or an aromatic polyanhydride. Representativepolyanhydrides include polyadipic anhydride, polyfumaric anhydride,polysebacic anhydride, polymaleic anhydride, polymalic anhydride,polyphthalic anhydride, polyisophthalic anhydride, polyasparticanhydride, polyterephthalic anhydride, polyisophthalic anhydride, polycarboxyphenoxypropane anhydride, polycarboxyphenoxyhexane anhydride, aswell as copolymers of these polyanhydrides with other polyanhydrides atdifferent mole ratios. Other suitable polyanhydrides are disclosed inU.S. Pat. Nos. 4,757,128, 4,857,311, 4,888,176, and 4,789,724. Thepolyanhydride can also be a copolymer containing polyanhydride blocks.

In certain embodiments, the hydrophobic polymer segment is polysebacicanhydride. In certain embodiments, the hydrophobic polymer segment ispoly(1,6-bis(p-carboxyphenoxy)hexane-co-sebacic acid) (poly(CPH-SA). Incertain embodiments, the hydrophobic polymer segment ispoly(1,3-bis(p-carboxyphenoxy)propane-co-sebacic acid) (poly(CPP-SA).

The molecular weight of the hydrophobic polymer can be varied to preparepolymer-drug conjugates that form particles having properties, such asdrug release rate, optimal for specific applications. The hydrophobicpolymer segment can have a molecular weight of about 150 Da to 1 MDa. Incertain embodiments, the hydrophobic polymer segment has a molecularweight of between about 1 kDa and about 100 kDa, more preferably betweenabout 1 kDa and about 50 kDa, most preferably between about 1 kDa andabout 25 kDa.

In some cases, the hydrophobic polymer segment has a molecular weightwhich is less that the average molecular weight of the one or morehydrophilic polymer segments of the polymer-drug conjugate. In apreferred embodiment, the hydrophobic polymer segment has a molecularweight of less than about 5 kDa.

D. Hydrophilic Polymers

Polymer-drug conjugates can also contain one or more hydrophilic polymersegments. Preferably, the polymer-drug conjugates contain more than onehydrophilic polymer segment. In some embodiments, the polymer-drugconjugate contains between two and six, more preferably between threeand five, hydrophilic polymer segments. In certain embodiments, thepolymer drug conjugate contains three hydrophilic polymer segments.

Each hydrophilic polymer segment can independently be any hydrophilic,biocompatible (i.e., it does not induce a significant inflammatory orimmune response), non-toxic polymer or copolymer. Examples of suitablepolymers include, but are not limited to, poly(alkylene glycols) such aspolyethylene glycol (PEG), poly(propylene glycol) (PPG), and copolymersof ethylene glycol and propylene glycol, poly(oxyethylated polyol),poly(olefinic alcohol), polyvinylpyrrolidone),poly(hydroxyalkylmethacrylamide), poly(hydroxyalkylmethacrylate),poly(saccharides), poly(amino acids), poly(hydroxy acids), poly(vinylalcohol), and copolymers, terpolymers, and mixtures thereof.

In preferred embodiments, the one or more hydrophilic polymer segmentscontain a poly(alkylene glycol) chain. The poly(alkylene glycol) chainsmay contain between 8 and 500 repeat units, more preferably between 40and 500 repeat units. Suitable poly(alkylene glycols) includepolyethylene glycol), polypropylene 1,2-glycol, poly(propylene oxide),polypropylene 1,3-glycol, and copolymers thereof. In certainembodiments, the one or more hydrophilic polymer segments are PEGchains. In such cases, the PEG chains can be linear or branched, such asthose described in U.S. Pat. No. 5,932,462. In certain embodiments, thePEG chains are linear.

Each of the one or more hydrophilic polymer segments can independentlyhave a molecular weight of about 300 Da to 1 MDa. The hydrophilicpolymer segment may have a molecular weight ranging between any of themolecular weights listed above. In certain embodiments, each of the oneor more hydrophilic polymer segments has a molecular weight of betweenabout 1 kDa and about 20 kDa, more preferably between about 1 kDa andabout 15 kDa, most preferably between about 1 kDa and about 10 kDa. In apreferred embodiment, each of the one or more hydrophilic polymersegments has a molecular weight of about 5 kDa.

E. Branch Points

The conjugates optionally contain a branch point which serves to connectmultiple hydrophilic polymer segments to one end of the hydrophobicpolymer segment. The branch point can be any organic, inorganic, ororganometallic moiety which is polyvalent, so as to provide more thantwo points of attachment. In preferred embodiments, the branch point isan organic molecule which contains multiple functional groups.

The functional groups may be any atom or group of atoms that contains atleast one atom that is neither carbon nor hydrogen, with the provisothat the groups must be capable of reacting with the hydrophobic andhydrophilic polymer segments. Suitable functional groups includehalogens (bromine, chlorine, and iodine); oxygen-containing functionalgroups such as a hydroxyls, epoxides, carbonyls, aldehydes, ester,carboxyls, and acid chlorides; nitrogen-containing functional groupssuch as amines and azides; and sulfur-containing groups such as thiols.The functional group may also be a hydrocarbon moiety which contains oneor more non-aromatic pi-bonds, such as an alkyne, alkene, or diene.Preferably, the branch point will contain at least two different typesof functional groups (e.g., one or more alcohols and one or morecarboxylic acids, or one or more halides and one or more alcohols). Insuch cases, the different functional groups present on the branch pointcan be independently addressed synthetically, permitting the covalentattachment of the hydrophobic and hydrophilic segments to the branchpoint in controlled stoichiometric ratios.

Following reaction of the hydrophobic and hydrophilic polymer segmentswith functional groups on the branch point, the one or more hydrophobicpolymer segments and the one or more hydrophilic polymer segments willbe covalently joined to the branch point via linking moieties. Theidentity of the linking moieties will be determined by the identity ofthe functional group and the reactive locus of the hydrophobic andhydrophilic polymer segments (as these elements react to form thelinking moiety or a precursor of the linking moiety). Examples ofsuitable linking moieties that connect the polymer segments to thebranch point include secondary amides (—CONH—), tertiary amides(—CONR—), secondary carbamates (—OCONH—; —NHCOO—), tertiary carbamates(—OCONR—; —NRCOO—), ureas (—NHCONH—; —NRCONH—; —NHCONR—, —NRCONR—),carbinols (—CHOH—, —CROH—), ethers (—O—), and esters (—COO—, —CH₂O₂C—,CHRO₂C—), wherein R is an alkyl group, an aryl group, or a heterocyclicgroup. In certain embodiments, the polymer segments are connected to thebranch point via an ester (—COO—, —CH₂O₂C—, CHRO₂C—), a secondary amide(—CONH—), or a tertiary amide (—CONR—), wherein R is an alkyl group, anaryl group, or a heterocyclic group.

In certain embodiments, the branch point is polycarboxylic acid, such ascitric acid, tartaric acid, mucic acid, gluconic acid, or5-hydroxybenzene-1,2,3,-tricarboxylic acid. Exemplary branch pointsinclude the following organic compounds:

F. Synthesis of Polymer-Drug Conjugates

Polymer-drug conjugates can be prepared using synthetic methods known inthe art. Representative methodologies for the preparation ofpolymer-drug conjugates are discussed below. The appropriate route forsynthesis of a given polymer-drug conjugate can be determined in view ofa number of factors, such as the structure of the polymer-drugconjugate, the identity of the polymers which make up the conjugate, theidentity of the active agent, as well as the structure of the compoundas a whole as it relates to compatibility of functional groups,protecting group strategies, and the presence of labile bonds.

In addition to the synthetic methodologies discussed below, alternativereactions and strategies useful for the preparation of the polymer-dugconjugates disclosed herein are known in the art. See, for example,March, “Advanced Organic Chemistry,” 5^(th) Edition, 2001,Wiley-Interscience Publication, New York).

Generally, polymer-drug conjugates are prepared by first forming thepolymeric component of the polymer-drug conjugate, and then covalentlyattaching an active agent. For example, Schemes 1 and 2 illustrate thesynthesis of a polymer-doxorubicin conjugate containing doxorubicinbound to a poly(sebacic anhydride) polymer segment to which is a singlepolyethylene glycol chain is attached (DXR-PSA-PEG).

In a first step, sebacic acid is refluxed is acetic anhydride to form anacylated polysebacic acid precursor (PreSA). An excess of PreSA is thencombined with polyethylene glycol methyl ether, and polymerized underanhydrous hot-melt polymerization conditions. As shown in Scheme 2, theresulting polymer (PEG-PSA) can then be reacted with doxorubicin to formthe polymer-drug conjugate (DXR-PSA-PEG).

The synthesis of an exemplary polymer-drug conjugate containing multiplehydrophilic polymer segments (three PEG chains) attached to ahydrophobic polymer segment (poly(sebacic anhydride) via a branch point(citric acid) is described in Schemes 3 and 4.

In the case of polymer-drug conjugates containing a branch point,synthesis of the polymer drug conjugate will typically begin bysequentially attaching the hydrophobic polymer segment and thehydrophilic polymer segments to the branch point to form the polymericportion of the polymer-drug conjugate. As shown in scheme 3, citric acidis first reacted with CH₃O-PEG-NH₂ in the presence ofN,N-dicyclohexylcarbodiimide (DCC) and a catalytic amount of4-dimethylaminopyridine (DMAP), forming amide linkages between the PEGchains and the three carboxylic acid residues of the citric acid branchpoint. The resulting compound is then reacted with an acylatedpolysebacic acid precursor (PreSA), and polymerized under anhydroushot-melt polymerization conditions. As shown in Scheme 4, he resultingpolymer (PEG₃-PSA) is then reacted with doxorubicin to form thepolymer-drug conjugate (DXR-PSA-PEG₃).

III. Particles and Implants for Controlled Delivery of HIF-1 Inhibitors

Polymeric implants (e.g., rods, discs, wafers, etc.), microparticles,and nanoparticles for the controlled delivery of one or more HIF-1inhibitors are provided, either formed of the conjugates or having theconjugates dispersed or encapsulated in a matrix. In some embodiments,the particles or implants contain one or more HIF-1 inhibitors dispersedor encapsulated in a polymeric matrix. In preferred embodiments, theparticles or implants are formed from polymer-drug conjugates containingone or more HIF-1 inhibitors that are covalently bound to a polymer.

A. Particles Formed from Polymer-Drug Conjugates

Microparticles and nanoparticles can be formed from one or morepolymer-drug conjugates. In some cases, particles are formed from asingle polymer-drug conjugate (i.e., the particles are formed from apolymer-drug conjugate which contains the same active agent, hydrophobicpolymer segment, branch point (when present), and hydrophilic polymersegment or segments).

In other embodiments, the particles are formed from a mixture of two ormore different polymer-drug conjugates. For example, particles may beformed from two or more polymer-drug conjugates containing differentHIF-1 inhibitors and the same hydrophobic polymer segment, branch point(when present), and hydrophilic polymer segment or segments. Suchparticles can be used, for example, to co-administer two or more HIF-1inhibitors. In other cases, the particles are formed from two or morepolymer-drug conjugates containing the same HIF-1 inhibitor, anddifferent hydrophobic polymer segments, branch points (when present),and/or hydrophilic polymer segments. Such particles can be used, forexample, to vary the release rate of HIF-1 inhibitors. The particles canalso be formed from two or more polymer-drug conjugates containingdifferent HIF-1 inhibitors and different hydrophobic polymer segments,branch points (when present), and/or hydrophilic polymer segments.

Particles can also be formed from blends of polymer-drug conjugates withone or more additional polymers. In these cases, the one or moreadditional polymers can be any of the non-biodegradable or biodegradablepolymers described in Section B below, although biodegradable polymersare preferred. In these embodiments, the identity and quantity of theone or more additional polymers can be selected, for example, toinfluence particle stability, i.e. that time required for distributionto the site where delivery is desired, and the time desired fordelivery.

Particles having an average particle size of between 10 nm and 1000microns are useful in the compositions described herein. In preferredembodiments, the particles have an average particle size of between 10nm and 100 microns, more preferably between about 100 nm and about 50microns, more preferably between about 200 nm and about 50 microns. Incertain embodiments, the particles are nanoparticles having a diameterof between 500 and 700 nm. The particles can have any shape but aregenerally spherical in shape.

In some embodiments, the population of particles formed from one or morepolymer-drug conjugates is a monodisperse population of particles. Inother embodiments, the population of particles formed from one or morepolymer-drug conjugates is a polydisperse population of particles. Insome instances where the population of particles formed from one or morepolymer-drug conjugates is polydisperse population of particles, greaterthat 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% of the particle sizedistribution lies within 10% of the median particle size.

Preferably, particles formed from one or more polymer-drug conjugatescontain significant amounts of a hydrophilic polymer, such as PEG, ontheir surface.

B. Particles Containing One or More HIF-1 Inhibitors Dispersed in aPolymer Matrix

Particles can also be formed containing one or more HIF-1 inhibitorsdispersed or encapsulated in a polymeric matrix. The matrix can beformed of non-biodegradable or biodegradable matrices, althoughbiodegradable matrices are preferred. The polymer is selected based onthe time required for in vivo stability, i.e. that time required fordistribution to the site where delivery is desired, and the time desiredfor delivery.

Representative synthetic polymers are: poly(hydroxy acids) such aspoly(lactic acid), poly(glycolic acid), and poly(lactic acid-co-glycolicacid), poly(lactide), poly(glycolide), poly(lactide-co-glycolide),polyanhydrides, polyorthoesters, polyamides, polycarbonates,polyalkylenes such as polyethylene and polypropylene, polyalkyleneglycols such as poly(ethylene glycol), polyalkylene oxides such aspoly(ethylene oxide), polyalkylene terephthalates such as poly(ethyleneterephthalate), polyvinyl alcohols, polyvinyl ethers, polyvinyl esters,polyvinyl halides such as poly(vinyl chloride), polyvinylpyrrolidone,polysiloxanes, poly(vinyl alcohols), poly(vinyl acetate), polystyrene,polyurethanes and co-polymers thereof, derivativized celluloses such asalkyl cellulose, hydroxyalkyl celluloses, cellulose ethers, celluloseesters, nitro celluloses, methyl cellulose, ethyl cellulose,hydroxypropyl cellulose, hydroxy-propyl methyl cellulose, hydroxybutylmethyl cellulose, cellulose acetate, cellulose propionate, celluloseacetate butyrate, cellulose acetate phthalate, carboxylethyl cellulose,cellulose triacetate, and cellulose sulphate sodium salt (jointlyreferred to herein as “synthetic celluloses”), polymers of acrylic acid,methacrylic acid or copolymers or derivatives thereof including esters,poly(methyl methacrylate), poly(ethyl methacrylate),poly(butylmethacrylate), poly(isobutyl methacrylate),poly(hexylmethacrylate), poly(isodecyl methacrylate), poly(laurylmethacrylate), poly(phenyl methacrylate), poly(methyl acrylate),poly(isopropyl acrylate), poly(isobutyl acrylate), and poly(octadecylacrylate) (jointly referred to herein as “polyacrylic acids”),poly(butyric acid), poly(valeric acid), andpoly(lactide-co-caprolactone), copolymers and blends thereof. As usedherein, “derivatives” include polymers having substitutions, additionsof chemical groups, for example, alkyl, alkylene, hydroxylations,oxidations, and other modifications routinely made by those skilled inthe art.

Examples of preferred biodegradable polymers include polymers of hydroxyacids such as lactic acid and glycolic acid, and copolymers with PEG,polyanhydrides, poly(ortho)esters, polyurethanes, poly(butyric acid),poly(valeric acid), poly(lactide-co-caprolactone), blends and copolymersthereof.

Examples of preferred natural polymers include proteins such as albuminand prolamines, for example, zein, and polysaccharides such as alginate,cellulose and polyhydroxyalkanoates, for example, polyhydroxybutyrate.The in vivo stability of the matrix can be adjusted during theproduction by using polymers such as polylactide co glycolidecopolymerized with polyethylene glycol (PEG). PEG if exposed on theexternal surface may elongate the time these materials circulate sinceit is hydrophilic.

Examples of preferred non-biodegradable polymers include ethylene vinylacetate, poly(meth)acrylic acid, polyamides, copolymers and mixturesthereof.

Particles having an average particle size of between 10 nm and 1000microns are useful in the compositions described herein. In preferredembodiments, the particles have an average particle size of between 10nm and 100 microns, more preferably between about 100 nm and about 50microns, more preferably between about 200 nm and about 50 microns. Incertain embodiments, the particles are nanoparticles having a diameterof between 500 and 700 nm. The particles can have any shape but aregenerally spherical in shape.

C. Methods of Forming Microparticles and Nanoparticles

Microparticle and nanoparticles can be formed using any suitable methodfor the formation of polymer micro- or nanoparticles known in the art.The method employed for particle formation will depend on a variety offactors, including the characteristics of the polymers present in thepolymer-drug conjugate or polymer matrix, as well as the desiredparticle size and size distribution. The type of HIF-1 inhibitor(s)being incorporated in the particles may also be a factor as some HIF-1inhibitors are unstable in the presence of certain solvents, in certaintemperature ranges, and/or in certain pH ranges.

In circumstances where a monodisperse population of particles isdesired, the particles may be formed using a method which produces amonodisperse population of nanoparticles. Alternatively, methodsproducing polydisperse nanoparticle distributions can be used, and theparticles can be separated using methods known in the art, such assieving, following particle formation to provide a population ofparticles having the desired average particle size and particle sizedistribution.

Common techniques for preparing microparticles and nanoparticlesinclude, but are not limited to, solvent evaporation, hot melt particleformation, solvent removal, spray drying, phase inversion, coacervation,and low temperature casting. Suitable methods of particle formulationare briefly described below. Pharmaceutically acceptable excipients,including pH modifying agents, disintegrants, preservatives, andantioxidants, can optionally be incorporated into the particles duringparticle formation.

1. Solvent Evaporation

In this method, the polymer-drug conjugate (or polymer matrix and one ormore HIF-1 inhibitors) is dissolved in a volatile organic solvent, suchas methylene chloride. The organic solution containing the polymer-drugconjugate is then suspended in an aqueous solution that contains asurface active agent such as poly(vinyl alcohol). The resulting emulsionis stirred until most of the organic solvent evaporated, leaving solidnanoparticles. The resulting nanoparticles are washed with water anddried overnight in a lyophilizer. Nanoparticles with different sizes andmorphologies can be obtained by this method.

Polymer-drug conjugates which contain labile polymers, such as certainpolyanhydrides, may degrade during the fabrication process due to thepresence of water. For these polymers, the following two methods, whichare performed in completely anhydrous organic solvents, can be used.

2. Hot Melt Particle Formation

In this method, the polymer-drug conjugate (or polymer matrix and one ormore HIF-1 inhibitors) is first melted, and then suspended in anon-miscible solvent (like silicon oil), and, with continuous stirring,heated to 5° C. above the melting point of the polymer-drug conjugate.Once the emulsion is stabilized, it is cooled until the polymer-drugconjugate particles solidify. The resulting nanoparticles are washed bydecantation with a suitable solvent, such as petroleum ether, to give afree-flowing powder. The external surfaces of particles prepared withthis technique are usually smooth and dense. Hot melt particle formationcan be used to prepare particles containing polymer-drug conjugateswhich are hydrolytically unstable, such as certain polyanhydrides.Preferably, the polymer-drug conjugate used to prepare microparticlesvia this method will have an overall molecular weight of less than75,000 Daltons.

3. Solvent Removal

Solvent removal can also be used to prepare particles from polymer-drugconjugates that are hydrolytically unstable. In this method, thepolymer-drug conjugate (or polymer matrix and one or more HIF-1inhibitors) is dispersed or dissolved in a volatile organic solvent suchas methylene chloride. This mixture is then suspended by stirring in anorganic oil (such as silicon oil) to form an emulsion. Solid particlesform from the emulsion, which can subsequently be isolated from thesupernatant. The external morphology of spheres produced with thistechnique is highly dependent on the identity of the polymer-drugconjugate.

4. Spray Drying

In this method, the polymer-drug conjugate (or polymer matrix and one ormore HIF-1 inhibitors) is dissolved in an organic solvent such asmethylene chloride. The solution is pumped through a micronizing nozzledriven by a flow of compressed gas, and the resulting aerosol issuspended in a heated cyclone of air, allowing the solvent to evaporatefrom the microdroplets, forming particles. Particles ranging between0.1-10 microns can be obtained using this method.

5. Phase Inversion

Particles can be formed from polymer-drug conjugates using a phaseinversion method. In this method, the polymer-drug conjugate (or polymermatrix and one or more HIF-1 inhibitors) is dissolved in a “good”solvent, and the solution is poured into a strong non solvent for thepolymer-drug conjugate to spontaneously produce, under favorableconditions, microparticles or nanoparticles. The method can be used toproduce nanoparticles in a wide range of sizes, including, for example,about 100 nanometers to about 10 microns, typically possessing a narrowparticle size distribution.

6. Coacervation

Techniques for particle formation using coacervation are known in theart, for example, in GB-B-929 406; GB-B-929 40 1; and U.S. Pat. Nos.3,266,987, 4,794,000, and 4,460,563. Coacervation involves theseparation of a polymer-drug conjugate (or polymer matrix and one ormore HIF-1 inhibitors) solution into two immiscible liquid phases. Onephase is a dense coacervate phase, which contains a high concentrationof the polymer-drug conjugate, while the second phase contains a lowconcentration of the polymer-drug conjugate. Within the dense coacervatephase, the polymer-drug conjugate forms nanoscale or microscaledroplets, which harden into particles.

Coacervation may be induced by a temperature change, addition of anon-solvent or addition of a micro-salt (simple coacervation), or by theaddition of another polymer thereby forming an interpolymer complex(complex coacervation).

7. Low Temperature Casting

Methods for very low temperature casting of controlled releasemicrospheres are described in U.S. Pat. No. 5,019,400 to Gombotz et al.In this method, the polymer-drug conjugate (or polymer matrix and one ormore HIF-1 inhibitors) is dissolved in a solvent. The mixture is thenatomized into a vessel containing a liquid non-solvent at a temperaturebelow the freezing point of the polymer-drug conjugate solution whichfreezes the polymer-drug conjugate droplets. As the droplets andnon-solvent for the polymer-drug conjugate are warmed, the solvent inthe droplets thaws and is extracted into the non-solvent, hardening themicrospheres.

D. Implants Formed from Polymer-Drug Conjugates

Implants can be formed from one or more polymer-drug conjugates. Inpreferred embodiments, the implants are intraocular implants. Suitableimplants include, but are not limited to, rods, discs, wafers, and thelike.

In some cases, the implants are formed from a single polymer-drugconjugate (i.e., the implants are formed from a polymer-drug conjugatewhich contains the same active agent, hydrophobic polymer segment,branch point (when present), and hydrophilic polymer segment orsegments).

In other embodiments, the implants are formed from a mixture of two ormore different polymer-drug conjugates. For example, implants may beformed from two or more polymer-drug conjugates containing differentHIF-1 inhibitors and the same hydrophobic polymer segment, branch point(when present), and hydrophilic polymer segment or segments. Suchimplants can be used, for example, to co-administer two or more HIF-1inhibitors. In other cases, the implants are formed from two or morepolymer-drug conjugates containing the same HIF-1 inhibitor, anddifferent hydrophobic polymer segments, branch points (when present),and/or hydrophilic polymer segments. Such implants can be used, forexample, to vary the release rate of HIF-1 inhibitors. The implants canalso be formed from two or more polymer-drug conjugates containingdifferent HIF-1 inhibitors and different hydrophobic polymer segments,branch points (when present), and/or hydrophilic polymer segments.

Implants can also be formed from a polymeric matrix having one or moreHIF-1 inhibitors dispersed or encapsulated therein. The matrix can beformed of any of the non-biodegradable or biodegradable polymersdescribed in Section B above, although biodegradable polymers arepreferred. The composition of the polymer matrix is selected based onthe time required for in vivo stability, i.e. that time required fordistribution to the site where delivery is desired, and the time desiredfor delivery.

Implants can also be formed from blends of polymer-drug conjugates withone or more of the polymers described in Section B above.

1. Implant Size and Shape

The implants may be of any geometry such as fibers, sheets, films,microspheres, spheres, circular discs, rods, or plaques. Implant size isdetermined by factors such as toleration for the implant, location ofthe implant, size limitations in view of the proposed method of implantinsertion, ease of handling, etc.

Where sheets or films are employed, the sheets or films will be in therange of at least about 0.5 mm×0.5 mm, usually about 3 to 10 mm×5 to 10mm with a thickness of about 0.1 to 1.0 mm for ease of handling. Wherefibers are employed, the fiber diameter will generally be in the rangeof about 0.05 to 3 mm and the fiber length will generally be in therange of about 0.5 to 10 mm.

The size and shape of the implant can also be used to control the rateof release, period of treatment, and drug concentration at the site ofimplantation. Larger implants will deliver a proportionately largerdose, but depending on the surface to mass ratio, may have a slowerrelease rate. The particular size and geometry of the implant are chosento suit the site of implantation.

Intraocular implants may be spherical or non-spherical in shape. Forspherical-shaped implants, the implant may have a largest dimension(e.g., diameter) between about 5 μm and about 2 mm, or between about 10μm and about 1 mm for administration with a needle, greater than 1 mm,or greater than 2 mm, such as 3 mm or up to 10 mm, for administration bysurgical implantation. If the implant is non-spherical, the implant mayhave the largest dimension or smallest dimension be from about 5 μm andabout 2 mm, or between about 10 μm and about 1 mm for administrationwith a needle, greater than 1 mm, or greater than 2 mm, such as 3 mm orup to 10 mm, for administration by surgical implantation.

The vitreous chamber in humans is able to accommodate relatively largeimplants of varying geometries, having lengths of, for example, 1 to 10mm. The implant may be a cylindrical pellet (e.g., rod) with dimensionsof about 2 mm×0.75 mm diameter. The implant may be a cylindrical pelletwith a length of about 7 mm to about 10 mm, and a diameter of about 0.75mm to about 1.5 mm. In certain embodiments, the implant is in the formof an extruded filament with a diameter of about 0.5 mm, a length ofabout 6 mm, and a weight of approximately 1 mg. In some embodiments, thedimension are, or are similar to, implants already approved forintraocular injection via needle: diameter of 460 microns and a lengthof 6 mm and diameter of 370 microns and length of 3.5 mm.

Intraocular implants may also be designed to be least somewhat flexibleso as to facilitate both insertion of the implant in the eye, such as inthe vitreous, and subsequent accommodation of the implant. The totalweight of the implant is usually about 250 to 5000 μg, more preferablyabout 500-1000 μg. In certain embodiments, the intraocular implant has amass of about 500 μg, 750 μg, or 1000 μg.

2. Methods of Manufacture

Implants can be manufactured using any suitable technique known in theart. Examples of suitable techniques for the preparation of implantsinclude solvent evaporation methods, phase separation methods,interfacial methods, molding methods, injection molding methods,extrusion methods, coextrusion methods, carver press method, die cuttingmethods, heat compression, and combinations thereof. Suitable methodsfor the manufacture of implants can be selected in view of many factorsincluding the properties of the polymer/polymer segments present in theimplant, the properties of the one or more HIF-1 inhibitors present inthe implant, and the desired shape and size of the implant. Suitablemethods for the preparation of implants are described, for example, inU.S. Pat. No. 4,997,652 and U.S. Patent Application Publication No. US2010/0124565.

In certain cases, extrusion methods may be used to avoid the need forsolvents during implant manufacture. When using extrusion methods, thepolymer/polymer segments and HIF-1 inhibitor are chosen so as to bestable at the temperatures required for manufacturing, usually at leastabout 85 degrees Celsius. However, depending on the nature of thepolymeric components and the one or more HIF-1 inhibitors, extrusionmethods can employ temperatures of about 25 degrees Celsius to about 150degrees Celsius, more preferably about 65 degrees Celsius to about 130degrees Celsius.

Implants may be coextruded in order to provide a coating covering all orpart of the surface of the implant. Such coatings may be erodible ornon-erodible, and may be impermeable, semi-permeable, or permeable tothe HIF-1 inhibitor, water, or combinations thereof. Such coatings canbe used to further control release of the HIF-1 inhibitor from theimplant.

Compression methods may be used to make the implants. Compressionmethods frequently yield implants with faster release rates thanextrusion methods. Compression methods may employ pressures of about50-150 psi, more preferably about 70-80 psi, even more preferably about76 psi, and use temperatures of about 0 degrees Celsius to about 115degrees Celsius, more preferably about 25 degrees Celsius.

IV. Pharmaceutical Formulations

Pharmaceutical formulations contain one or more polymer-drug conjugatesin combination with one or more pharmaceutically acceptable excipients.Representative excipients include solvents, diluents, pH modifyingagents, preservatives, antioxidants, suspending agents, wetting agents,viscosity modifiers, tonicity agents, stabilizing agents, andcombinations thereof. Suitable pharmaceutically acceptable excipientsare preferably selected from materials which are generally recognized assafe (GRAS), and may be administered to an individual without causingundesirable biological side effects or unwanted interactions.

In some cases, the pharmaceutical formulation contains only one type ofconjugate or polymeric particles for the controlled release of HIF-1inhibitors (e.g., a formulation containing polymer-drug conjugateparticles wherein the polymer-drug conjugate particles incorporated intothe pharmaceutical formulation have the same composition). In otherembodiments, the pharmaceutical formulation contains two or moredifferent type of conjugates or polymeric particles for the controlledrelease of HIF-1 inhibitors (e.g., the pharmaceutical formulationcontains two or more populations of polymer-drug conjugate particles,wherein the populations of polymer-drug conjugate particles havedifferent chemical compositions, different average particle sizes,and/or different particle size distributions).

A. Additional Active Agents

In addition to the one or more HIF-1 inhibitors present in the polymericparticles, the formulation can contain one or more additionaltherapeutic, diagnostic, and/or prophylactic agents. The active agentscan be a small molecule active agent or a biomolecule, such as an enzymeor protein, polypeptide, or nucleic acid. Suitable small molecule activeagents include organic and organometallic compounds. In some instances,the small molecule active agent has a molecular weight of less thanabout 2000 g/mol, more preferably less than about 1500 g/mol, mostpreferably less than about 1200 g/mol. The small molecule active agentcan be a hydrophilic, hydrophobic, or amphiphilic compound.

In some cases, one or more additional active agents may be encapsulatedin, dispersed in, or otherwise associated with particles formed from oneor more polymer-drug conjugates. In certain embodiments, one or moreadditional active agents may also be dissolved or suspended in thepharmaceutically acceptable carrier.

In the case of pharmaceutical compositions for the treatment of oculardiseases, the formulation may contain one or more ophthalmic drugs. Inparticular embodiments, the ophthalmic drug is a drug used to treat,prevent or diagnose a disease or disorder of the posterior segment eye.Non-limiting examples of ophthalmic drugs include anti-glaucoma agents,anti-angiogenesis agents, anti-infective agents, anti-inflammatoryagents, growth factors, immunosuppressant agents, anti-allergic agents,and combinations thereof.

Representative anti-glaucoma agents include prostaglandin analogs (suchas travoprost, bimatoprost, and latanoprost), beta-andrenergic receptorantagonists (such as timolol, betaxolol, levobetaxolol, and carteolol),alpha-2 adrenergic receptor agonists (such as brimonidine andapraclonidine), carbonic anhydrase inhibitors (such as brinzolamide,acetazolamine, and dorzolamide), miotics (i.e., parasympathomimetics,such as pilocarpine and ecothiopate), seretonergics muscarinics,dopaminergic agonists, and adrenergic agonists (such as apraclonidineand brimonidine).

Representative anti-angiogenesis agents include, but are not limited to,antibodies to vascular endothelial growth factor (VEGF) such asbevacizumab (AVASTIN®) and rhuFAb V2 (ranibizumab, LUCENTIS®), and otheranti-VEGF compounds including aflibercept (EYLEA®); MACUGEN® (pegaptanimsodium, anti-VEGF aptamer or EYE001) (Eyetech Pharmaceuticals); pigmentepithelium derived factor(s) (PEDF); COX-2 inhibitors such as celecoxib(CELEBREX®) and rofecoxib (VIOXX®); interferon alpha; interleukin-12(IL-12); thalidomide (THALOMID®) and derivatives thereof such aslenalidomide (REVLIMID®); squalamine; endostatin; angiostatin; ribozymeinhibitors such as ANGIOZYME® (Sirna Therapeutics); multifunctionalantiangiogenic agents such as NEOVASTAT® (AE-941) (Aeterna Laboratories,Quebec City, Canada); receptor tyrosine kinase (RTK) inhibitors such assunitinib (SUTENT®); tyrosine kinase inhibitors such as sorafenib(Nexavar®) and erlotinib (Tarceva®); antibodies to the epidermal grownfactor receptor such as panitumumab (VECTIBIX®) and cetuximab(ERBITUX®), as well as other anti-angiogenesis agents known in the art.

Anti-infective agents include antiviral agents, antibacterial agents,antiparasitic agents, and anti-fungal agents. Representative antiviralagents include ganciclovir and acyclovir. Representative antibioticagents include aminoglycosides such as streptomycin, amikacin,gentamicin, and tobramycin, ansamycins such as geldanamycin andherbimycin, carbacephems, carbapenems, cephalosporins, glycopeptidessuch as vancomycin, teicoplanin, and telavancin, lincosamides,lipopeptides such as daptomycin, macrolides such as azithromycin,clarithromycin, dirithromycin, and erythromycin, monobactams,nitrofurans, penicillins, polypeptides such as bacitracin, colistin andpolymyxin B, quinolones, sulfonamides, and tetracyclines.

In some cases, the active agent is an anti-allergic agent such asolopatadine and epinastine.

Anti-inflammatory agents include both non-steroidal and steroidalanti-inflammatory agents. Suitable steroidal active agents includeglucocorticoids, progestins, mineralocorticoids, and corticosteroids.

The ophthalmic drug may be present in its neutral form, or in the formof a pharmaceutically acceptable salt. In some cases, it may bedesirable to prepare a formulation containing a salt of an active agentdue to one or more of the salt's advantageous physical properties, suchas enhanced stability or a desirable solubility or dissolution profile.

Generally, pharmaceutically acceptable salts can be prepared by reactionof the free acid or base forms of an active agent with a stoichiometricamount of the appropriate base or acid in water or in an organicsolvent, or in a mixture of the two; generally, non-aqueous media likeether, ethyl acetate, ethanol, isopropanol, or acetonitrile arepreferred. Pharmaceutically acceptable salts include salts of an activeagent derived from inorganic acids, organic acids, alkali metal salts,and alkaline earth metal salts as well as salts formed by reaction ofthe drug with a suitable organic ligand (e.g., quaternary ammoniumsalts). Lists of suitable salts are found, for example, in Remington'sPharmaceutical Sciences, 20th ed., Lippincott Williams & Wilkins,Baltimore, Md., 2000, p. 704. Examples of ophthalmic drugs sometimesadministered in the form of a pharmaceutically acceptable salt includetimolol maleate, brimonidine tartrate, and sodium diclofenac.

In some cases, the active agent is a diagnostic agent imaging orotherwise assessing the eye. Exemplary diagnostic agents includeparamagnetic molecules, fluorescent compounds, magnetic molecules, andradionuclides, x-ray imaging agents, and contrast media.

In certain embodiments, the pharmaceutical composition contains one ormore local anesthetics. Representative local anesthetics includetetracaine, lidocaine, amethocaine, proparacaine, lignocaine, andbupivacaine. In some cases, one or more additional agents, such as ahyaluronidase enzyme, is also added to the formulation to accelerate andimproves dispersal of the local anesthetic.

B. Formulations for Ocular Administration

Particles formed from the polymer-drug conjugates will preferably beformulated as a solution or suspension for injection to the eye.

Pharmaceutical formulations for ocular administration are preferably inthe form of a sterile aqueous solution or suspension of particles formedfrom one or more polymer-drug conjugates. Acceptable solvents include,for example, water, Ringer's solution, phosphate buffered saline (PBS),and isotonic sodium chloride solution. The formulation may also be asterile solution, suspension, or emulsion in a nontoxic, parenterallyacceptable diluent or solvent such as 1,3-butanediol.

In some instances, the formulation is distributed or packaged in aliquid form. Alternatively, formulations for ocular administration canbe packed as a solid, obtained, for example by lyophilization of asuitable liquid formulation. The solid can be reconstituted with anappropriate carrier or diluent prior to administration.

Solutions, suspensions, or emulsions for ocular administration may bebuffered with an effective amount of buffer necessary to maintain a pHsuitable for ocular administration. Suitable buffers are well known bythose skilled in the art and some examples of useful buffers areacetate, borate, carbonate, citrate, and phosphate buffers.

Solutions, suspensions, or emulsions for ocular administration may alsocontain one or more tonicity agents to adjust the isotonic range of theformulation. Suitable tonicity agents are well known in the art and someexamples include glycerin, mannitol, sorbitol, sodium chloride, andother electrolytes.

Solutions, suspensions, or emulsions for ocular administration may alsocontain one or more preservatives to prevent bacterial contamination ofthe ophthalmic preparations. Suitable preservatives are known in theart, and include polyhexamethylenebiguanidine (PHMB), benzalkoniumchloride (BAK), stabilized oxychloro complexes (otherwise known asPurite®), phenylmercuric acetate, chlorobutanol, sorbic acid,chlorhexidine, benzyl alcohol, parabens, thimerosal, and mixturesthereof.

Solutions, suspensions, or emulsions for ocular administration may alsocontain one or more excipients known art, such as dispersing agents,wetting agents, and suspending agents.

V. Methods of Use

Controlled release dosage formulations for the delivery of one or moreHIF-1 inhibitors can be used to treat or a disease or disorder in apatient associated with vascularization, including cancer and obesity.In preferred embodiment, the pharmaceutical compositions areadministered to treat or prevent a disease or disorder in a patientassociated with ocular neovascularization. Upon administration, the oneor more HIF-1 inhibitors are released over an extended period of time atconcentrations which are high enough to produce therapeutic benefit, butlow enough to avoid cytotoxicity.

When administered to the eye, the particles release a low dose of one ormore active agents over an extended period of time, preferably longerthan 3, 7, 10, 15, 21, 25, 30, or 45 days. The structure of thepolymer-drug conjugate or makeup of the polymeric matrix, particlemorphology, and dosage of particles administered can be tailored toadminister a therapeutically effective amount of one or more activeagents to the eye over an extended period of time while minimizing sideeffects, such as the reduction of scoptopic ERG b-wave amplitudes and/orretinal degeneration.

A. Diseases and Disorders of the Eye

Pharmaceutical compositions containing particles for the controlledrelease of one or more HIF-1 inhibitors can be administered to the eyeof a patient in need thereof to treat or prevent one or more diseases ordisorders of the eye. In some cases, the disease or disorder of the eyeaffects the posterior segment of the eye. The posterior segment of theeye, as used herein, refers to the back two-thirds of the eye, includingthe anterior hyaloid membrane and all of the optical structures behindit, such as the vitreous humor, retina, choroid, and optic nerve.

In preferred embodiments, a pharmaceutical composition containingparticles formed from one or more of the polymer-drug conjugatesprovided herein is administered to treat or prevent an intraocularneovascular disease. In certain embodiments, the particles are formedfrom a polymer-drug conjugate containing an anthracycline, such asdaunorubicin or doxorubicin.

Eye diseases, particularly those characterized by ocularneovascularization, represent a significant public health concern.Intraocular neovascular diseases are characterized by unchecked vasculargrowth in one or more regions of the eye. Unchecked, the vascularizationdamages and/or obscures one or more structures in the eye, resulting invision loss. Intraocular neovascular diseases include proliferativeretinopathies, choroidal neovascularization (CNV), age-related maculardegeneration (AMD), diabetic and other ischemia-related retinopathies,diabetic macular edema, pathological myopia, von Hippel-Lindau disease,histoplasmosis of the eye, central retinal vein occlusion (CRVO),corneal neovascularization, and retinal neovascularization (RNV).Intraocular neovascular diseases afflict millions worldwide, in manycases leading to severe vision loss and a decrease in quality of lifeand productivity.

Age related macular degeneration (AMD) is a leading cause of severe,irreversible vision loss among the elderly. Bressler, et al. JAMA,291:1900-1901 (2004). AMD is characterized by a broad spectrum ofclinical and pathologic findings, such as pale yellow spots known asdrusen, disruption of the retinal pigment epithelium (RPE), choroidalneovascularization (CNV), and disciform macular degeneration. AMD isclassified as either dry (i.e., non-exudative) or wet (i.e., exudative).Dry AMD is characterized by the presence of lesions called drusen. WetAMD is characterized by neovascularization in the center of the visualfield.

Although less common, wet AMD is responsible for 80%-90% of the severevisual loss associated with AMD (Ferris, et al. Arch. Ophthamol.102:1640-2 (1984)). The cause of AMD is unknown. However, it is clearthat the risk of developing AMD increases with advancing age. AMD hasalso been linked to risk factors including family history, cigarettesmoking, oxidative stress, diabetes, alcohol intake, and sunlightexposure.

Wet AMD is typically characterized by CNV of the macular region. Thechoroidal capillaries proliferate and penetrate Bruch's membrane toreach the retinal pigment epithelium (RPE). In some cases, thecapillaries may extend into the subretinal space. The increasedpermeability of the newly formed capillaries leads to accumulation ofserous fluid or blood under the RPE and/or under or within theneurosensory retina. Decreases in vision occur when the fovea becomesswollen or detached. Fibrous metaplasia and organization may ensue,resulting in an elevated subretinal mass called a disciform scar thatconstitutes end-stage AMD and is associated with permanent vision loss(D'Amico D J. N. Engl. J. Med. 331:95-106 (1994)).

Other diseases and disorders of the eye, such as uveitis, are alsodifficult to treat using existing therapies. Uveitis is a general termreferring to inflammation of any component of the uveal tract, such asthe iris, ciliary body, or choroid. Inflammation of the overlyingretina, called retinitis, or of the optic nerve, called optic neuritis,may occur with or without accompanying uveitis.

Ocular complications of uveitis may produce profound and irreversibleloss of vision, especially when unrecognized or treated improperly. Themost frequent complications of uveitis include retinal detachment,neovascularization of the retina, optic nerve, or iris, and cystoidmacular edema. Macular edema (ME) can occur if the swelling, leaking,and background diabetic retinopathy (BDR) occur within the macula, thecentral 5% of the retina most critical to vision. ME is a common causeof severe visual impairment.

There have been many attempts to treat intraocular neurovasculardiseases, as well as diseases associated with chronic inflammation ofthe eye, with pharmaceuticals. Attempts to develop clinically usefultherapies have been plagued by difficulty in administering andmaintaining a therapeutically effective amount of the pharmaceutical inthe ocular tissue for an extended period of time. In addition, manypharmaceuticals exhibit significant side effects and/or toxicity whenadministered to the ocular tissue.

Intraocular neovascular diseases are diseases or disorders of the eyethat are characterized by ocular neovascularization. Theneovascularization may occur in one or more regions of the eye,including the cornea, retina, choroid layer, or iris. In certaininstances, the disease or disorder of the eye is characterized by theformation of new blood vessels in the choroid layer of the eye (i.e.,choroidal neovascularization, CNV). In some instances, the disease ordisorder of the eye is characterized by the formation of blood vesselsoriginating from the retinal veins and extending along the inner(vitreal) surface of the retina (i.e., retinal neovascularization, RNV).

Exemplary neovascular diseases of the eye include age-related maculardegeneration associated with choroidal neovascularization, proliferativediabetic retinopathy (diabetic retinopathy associated with retinal,preretinal, or iris neovascularization), proliferativevitreoretinopathy, retinopathy of prematurity, pathological myopia, vonHippel-Lindau disease, presumed ocular histoplasmosis syndrome (POHS),and conditions associated with ischemia such as branch retinal veinocclusion, central retinal vein occlusion, branch retinal arteryocclusion, and central retinal artery occlusion.

The neovascularization can be caused by a tumor. The tumor may be eithera benign or malignant tumor. Exemplary benign tumors include hamartomasand neurofibromas. Exemplary malignant tumors include choroidalmelanoma, uveal melanoma or the iris, uveal melanoma of the ciliarybody, retinoblastoma, or metastatic disease (e.g., choroidalmetastasis).

The neovascularization may be associated with an ocular wound. Forexample, the wound may the result of a traumatic injury to the globe,such as a corneal laceration. Alternatively, the wound may be the resultof ophthalmic surgery.

The polymer-drug conjugates can be administered to prevent or reduce therisk of proliferative vitreoretinopathy following vitreoretinal surgery,prevent corneal haze following corneal surgery (such as cornealtransplantation and eximer laser surgery), prevent closure of atrabeculectomy, or to prevent or substantially slow the recurrence ofpterygii.

The polymer-drug conjugates can be administered to treat or prevent aneye disease associated with inflammation. In such cases, thepolymer-drug conjugate preferably contains an anti-inflammatory agent.Exemplary inflammatory eye diseases include, but are not limited to,uveitis, endophthalmitis, and ophthalmic trauma or surgery.

The eye disease may also be an infectious eye disease, such as HIVretinopathy, toxocariasis, toxoplasmosis, and endophthalmitis.

Pharmaceutical compositions containing particles formed from one or moreof the polymer-drug conjugates can also be used to treat or prevent oneor more diseases that affect other parts of the eye, such as dry eye,meibomitis, glaucoma, conjunctivitis (e.g., allergic conjunctivitis,vernal conjunctivitis, giant papillary conjunctivitis, atopickeratoconjunctivitis), neovascular glaucoma with irisneovascularization, and iritis.

1. Methods of Administration

a. Mode of Administration

The formulations described herein can be administered locally to the eyeby intravitreal injection (e.g., front, mid or back vitreal injection),subconjunctival injection, intracameral injection, injection into theanterior chamber via the temporal limbus, intrastromal injection,injection into the subchoroidal space, intracorneal injection,subretinal injection, and intraocular injection. In a preferredembodiment, the pharmaceutical composition is administered byintravitreal injection.

The implants described herein can be administered to the eye usingsuitable methods for implantation known in the art. In certainembodiments, the implants are injected intravitreally using a needle,such as a 22-guage needle. Placement of the implant intravitreally maybe varied in view of the implant size, implant shape, and the disease ordisorder to be treated.

In some embodiments, the pharmaceutical compositions and/or implantsdescribed herein are co-administered with one or more additional activeagents. “Co-administration”, as used herein, refers to administration ofthe controlled release formulation of one or more HIF-1 inhibitors withone or more additional active agents within the same dosage form, aswell as administration using different dosage forms simultaneously or asessentially the same time. “Essentially at the same time” as used hereingenerally means within ten minutes, preferably within five minutes, morepreferably within two minutes, most preferably within in one minute.

In some embodiments, the pharmaceutical compositions and/or implantsdescribed herein are co-administered with one or more additionaltreatments for a neovascular disease or disorder of the eye. In someembodiments, the pharmaceutical compositions and/or implants describedherein are co-administered with one or more anti-angiogenesis agent suchbevacizumab (AVASTIN®), ranibizumab, LUCENTIS®, or aflibercept (EYLEA®).

b. Dosage

Preferably, the particles will release an effective amount of one ormore HIF-1 inhibitors over an extended period of time. In preferredembodiments, the particles release an effective amount of one or moreHIF-1 inhibitors over a period of at least two weeks, more preferablyover a period of at least four weeks, more preferably over a period ofat least six weeks, most preferably over a period of at least eightweeks. In some embodiments, the particles release an effective amount ofone or more HIF-1 inhibitors over a period of three months or longer.

In some cases, a pharmaceutical formulation is administered to a patientin need thereof in a therapeutically effective amount to decreasechoroidal neovascularization. In some cases, a pharmaceuticalformulation is administered to a patient in need thereof in atherapeutically effective amount to decrease retinal neovascularization.

c. Therapeutic Efficacy

In the case of age-related macular degeneration, therapeutic efficacy ina patient can be measured by one or more of the following: assessing themean change in the best corrected visual acuity (BCVA) from baseline toa desired time, assessing the proportion of patients who lose fewer than15 letters (three lines) in visual acuity at a desired time as comparedto a baseline, assessing the proportion of patients who gain greaterthan or equal to 15 letters (three lines) in visual acuity at a desiredtime as compared to a baseline, assessing the proportion of patientswith a visual acuity Snellen equivalent of 20/2000 or worse at a desiredtime, assessing the National Eye Institute Visual FunctioningQuestionnaire, and assessing the size of CNV and the amount of leakageof CNV at a desired time using fluorescein angiography.

In certain embodiments, at least 25%, more preferably at least 30%, morepreferably at least 35%, most preferably at least 40% of the patientswith recent onset CNV who are treated with the formulations describedherein improve by three or more lines of vision.

B. Other Diseases and Disorders

Controlled release dosage formulations for the delivery of one or moreHIF-1 inhibitors can be used to treat or a disease or disorder in apatient associated with vascularization, including cancer and obesity.

The present invention will be further understood by reference to thefollowing non-limiting examples.

EXAMPLES Example 1 Preparation of Polyanhydride-Drug Conjugate ParticlesSynthesis of Polymer

(Polyethylene glycol)₃-co-poly(sebacic acid) (PEG₃-PSA) was prepared bymelt polycondensation. Briefly, sebacic acid was refluxed in aceticanhydride to form a sebacic acid prepolymer (Acyl-SA).Citric-Polyethylene glycol (PEG₃) was prepared using methods known inthe art (Ben-Shabat, S. et al. Macromol. Biosci. 6:1019-1025 (2006)).2.0 g of CH₃O-PEG-NH₂, 26 mg of citric acid, 83 mg ofdicyclohexylcarbodiimide (DCC), and 4.0 mg of 4-(dimethylamino)pyridine(DMAP) were added to 10 mL of methylene chloride. This mixture wasstirred overnight at room temperature, then precipitated, washed withether, and dried under vacuum to isolate PEG₃. Next, Acyl-SA (90% w/w)and PEG₃ (10% w/w) were polymerized at 180° C. for 30 minutes. Nitrogengas was swept into the flask for 30 seconds every 15 minutes. Thereaction was allowed to proceed for 30 min. Polymers were cooled toambient temperature, dissolved in chloroform, and precipitated intoexcess petroleum ether. The precipitate was collected by filtration anddried under vacuum to constant weight.

Formation of DXR-PSA-PEG₃ Nanoparticles

DXR-PSA-PEG₃ nanoparticles were prepared by dissolving PEG₃-PSA with DXRat defined ratios in 3 mL dichloromethane and 1 mL DMSO and reacting for2 hrs at 50° before homogenizing (L4RT, Silverson Machines, EastLongmeadow, Mass.) into 100 mL of an aqueous solution containing 1%polyvinyl alcohol (25 kDa, Sigma). Particles were then hardened byallowing chloroform to evaporate at room temperature while stirring for2 hours. The particles were collected by centrifugation (20,000×g for 20min at 4° C.), and washed thrice with double distilled water. Particlesize was determined by dynamic light scattering using a ZetaSizer NanoZS (Malvern Instruments, Southborough, Mass.). Size measurements wereperformed at 25° C. at a scattering angle of 90°.

DXR Release from DXR-PSA-PEG₃ Nanoparticles In Vitro

DXR-PSA-PEG₃ nanoparticles were suspended in phosphate buffered saline(PBS, pH 7.4) at 2 mg/mL and incubated at 37° C. on a rotating platform(140 RPM). At selected time points, supernatant was collected bycentrifugation (13,500×g for 5 min) and particles were resuspended infresh PBS. DXR content was measured by absorbance at 480 nm.

Results

The DXR-PSA-PEG₃ nanoparticles prepared above contained 23.6% DXR (byweight), and had an average particle size of 647 nm. In vitro studiesshowed that DXR was released from the nanoparticles as a conjugate withsebacic acid in a steady fashion for up to two weeks under sinkconditions in PBS at 37° C. with no initial rapid drug release phase(i.e., no “burst effect”).

Example 2 Treatment of Choroidal Neovascularization in a Mouse Model ofCNV

Materials and Methods

Pathogen-free C57BL/6 mice (Charles River, Wilmington, Mass.) weretreated in accordance with the Association for Research in Vision and

Ophthalmology Statement for the Use of Animals in Ophthalmic and VisionResearch and the guidelines of the Johns Hopkins University Animal Careand Use Committee.

Choroidal NV was induced by laser photocoagulation-induced rupture ofBruch's membrane as previously described (To be, T. et al., Am. J.Pathol. 135(5): 1641-1646 (1998)). Briefly, 5-6-week-old female C57BL/6mice were anesthetized with ketamine hydrochloride (100 mg/kg bodyweight) and pupils were dilated. Laser photocoagulation (75 μm spotsize, 0.1 sec duration, 120 mW) was performed in the 9, 12, and 3o'clock positions of the posterior pole of each eye with the slit lampdelivery system of an OcuLight GL diode laser (Iridex, Mountain View,Calif.) and a handheld cover slip as a contact lens to view the retina.Production of a bubble at the time of laser, which indicates rupture ofBruch's membrane, is an important factor in obtaining CNV; therefore,only burns in which a bubble was produced were included in the study.

Immediately after laser-induced rupture of Bruch's membrane, mice wererandomized to various treatment groups for intraocular injections.Intravitreal injections were done under a dissecting microscope with aHarvard Pump Microinjection System and pulled glass micropipettes.

At 1, 4, 7, and 14 days after injection, fundus photographs were takenwith a Micron III® camera (Phoenix Research Laboratories Inc.,Pleasanton, Calif.). After 14 days, the mice were perfused with 1 ml ofPBS containing 25 mg/ml of fluorescein-labeled dextran (2×10⁶ Daltonsaverage molecular weight; Sigma-Aldrich, St. Louis, Mo.) and choroidalflat mounts were examined by fluorescence microscopy. Images werecaptured with a Nikon Digital Still Camera DXM1200 (Nikon InstrumentsInc., New York, N.Y.). Image analysis software (Image-Pro® Plus; MediaCybernetics, Silver Spring, Md.) was used to measure the total area ofCNV at each rupture site with the investigator masked with respect totreatment group.

Treatment of Oxygen-Induced Ischemic Retinopathy

C57BL/6 mice placed in 75% oxygen at postnatal day (P) 7 and at P12 werereturned to room air and given an intraocular injection of PBS or PBScontaining Daunorubicin, Doxorubicin, or DXR-PSA-PEG₃ nanoparticles. AtP17, the area of retinal NV on the surface of the retina was measured.Briefly, P17 mice were given an intraocular injection of 1 μl of ratanti-mouse platelet endothelial cell adhesion molecule-1 (PECAM-1)antibody (Pharmingen, San Jose, Calif.) and after 12 hours they wereeuthanized and eyes were fixed in PBS-buffered formalin for 5 hours atroom temperature. Retinas were dissected, washed, and incubated withgoat-anti rat polyclonal antibody conjugated with Alexa 488 (Invitrogen,Carlsbad, Calif.) at 1:500 dilution at room temperature for 45 minutesand flat mounted. An observer masked with respect to treatment groupmeasured the area of NV per retina by image analysis.

Treatment of VEGF-Induced Retinal Neovascularization

Hemizygous rhodopsin/VEGF transgenic mice that express VEGF inphotoreceptors were given an intraocular injection of 1 μl of PBS or PBScontaining 10 ug DXR-PSA-PEG₃ nanoparticles at P14. At P21, P28, P35,P42 or P49, the mice were anesthetized, perfused withfluorescein-labeled dextran (2×10⁶ average molecular weight,Sigma-Aldrich), and retinal flat mounts were examined by fluorescencemicroscopy (Axioskop2 plus; Zeiss, Thornwood, N.Y.) at 400×magnification, which provides a narrow depth of field, so that whenneovascularization along the outer edge of the retina is brought intofocus, the remainder of the retinal vessels are out of focus, allowingeasy delineation and quantification of the neovascularization. Imageswere digitized with a three-color charge-coupled device video camera(Cool SNAPTM-Pro; Media Cybernetics, Silver Spring, Md.) and a framegrabber. Image analysis software (Image-Pro Plus 5.0; Media Cybernetics,Silver Spring, Md.) was set to recognize fluorescently stainedneovascularization and used to calculate the total area ofneovascularization per retina. The investigator performing imageanalysis was masked with respect to treatment group.

Recording Of Electroretinograms (ERGs)

Adult C57BL/6 mice were given an intraocular injection of 1 μl of PBS orPBS containing of 0.1, 1.0, or 10 μg of Daunorubicin or Doxorubicin, or1.0 or 10 μg DXR-PSA-PEG₃ nanoparticles. Scotopic and photopic ERGs wererecorded at one, seven and 14 days after injection using an Espion ERGDiagnosys machine. For scotopic recordings, mice were dark adaptedovernight, and for photopic recordings, mice were adapted for 10 min tobackground white light at an intensity of 30 cd/m². The mice wereanesthetized with an intraperitoneal injection of ketamine hydrochloride(100 mg/kg body weight) and xylazine (5 mg/kg body weight). Pupils weredilated with Midrin P containing of 0.5% tropicamide and 0.5%phenylephrine, hydrochloride (Santen Pharmaceutical Co., Osaka, Japan).The mice were placed on a pad heated to 39° C. and platinum loopelectrodes were placed on each cornea after application of Gonioscopicprism solution (Alcon Labs, Fort Worth, Tex.). A reference electrode wasplaced subcutaneously in the anterior scalp between the eyes and aground electrode was inserted into the tail. The head of the mouse washeld in a standardized position in a ganzfeld bowl illuminator thatensured equal illumination of the eyes. Recordings for both eyes weremade simultaneously with electrical impedance balanced. Scotopic ERGswere recorded at 11 intensity levels of white light ranging from −3.00to 1.40 log cd-s/m2. Six measurements were averaged for each flashintensity. Photopic ERGs were recorded at three intensity levels ofwhite light ranging from 0.60 to 1.40 log cd-s/m2 with a 30 cd/m2background. Five measurements were averaged for each flash intensity.

Measurement of Outer Nuclear Layer (ONL) Thickness

ONL thickness was measured. Adult C57BL/6 mice were given an intraocularinjection of 1 μl of PBS or PBS containing of 0.1, 1.0, or 10 μg ofDaunorubicin or Doxorubicin, or 1.0 or 10 μg DXR-PSA-PEG₃ nanoparticles.Mice were euthanized, a mark was placed at 12:00 at the corneal limbus,and eyes were removed and embedded in optimal cutting temperaturecompound. Ten micrometer frozen sections were cut parallel to the 12:00or 9:00 meridian through the optic nerve and fixed in 4%paraformaldehyde. The sections were stained with hematoxylin and eosin,examined with an Axioskop microscope (Zeiss, Thornwood, N.Y.), andimages were digitized using a three charge-coupled device (CCD) colorvideo camera (IK-TU40A; Toshiba, Tokyo, Japan) and a frame grabber.Image-Pro Plus software (Media Cybernetics, Silver Spring, Md.) was usedto outline the ONL. With the observer masked with respect to treatmentgroup, ONL thickness was measured at six locations, 25% (51), 50% (S2),and 75% (S3) of the distance between the superior pole and the opticnerve and 25% (I1), 50% (I2), and 75% (I3) of the distance between theinferior pole the optic nerve.

Statistical Analysis

Data were expressed as mean±SEM. Statistical analysis was performedusing Student's t-test and P<0.05 was considered significant.

Results

Anthracyclines Suppress Choroidal and Retinal NV

In a mouse model of choroidal NV (To be, T. et al. Am. J. Pathol.153:1641-1646 (1998)) that is predictive of drug effects in patientswith neovascular AMD (Saishin, Y. et al. J. Cell Physiol. 195:241-248(2003)), intraocular injection of 10 μg of DNR suppressed choroidal NV,while injection of 1 or 0.1 μg had no significant effect (FIG. 1A).Similarly, intraocular injection of 10 μg of DXR suppressed choroidal NVand injections of 1 or 0.1 μg did not have a significant effect (FIG.1B).

In neonatal mice with oxygen-induced ischemic retinopathy, a modelpredictive of effects in proliferative diabetic retinopathy, intraocularinjection of 1 μg of DNR markedly reduced the area of retinal NV, 0.1 μgcaused a small reduction, and 0.01 μg had no significant effect (FIG.2A). The NV was visualized on retinal flat mounts after in vivoimmunofluorescent staining with anti-PECAM1, a technique thatselectively stains NV and hyaloid vessels. Intraocular injection of 1 μgof DXR, but not 0.1 or 0.01 μg, significantly reduced the area ofretinal NV (FIG. 2B). Five days after injection of 1 μg of DNR or DXR,precipitated drug was visualized on the surface of the retina. The meanarea of choroidal or retinal NV in fellow eyes was not significantlydifferent from that in eyes of mice in which both eyes were injectedwith vehicle indicating that there was no systemic effect fromintraocular injections of DNR or DXR.

Effect of Intraocular Injections of DNR or DXR on Retinal Function

Since DNR and DXR are antimetabolites as well as HIF-1 inhibitors weexamined their effect on retinal function assessed by ERGs. Fourteendays after intraocular injections of 1 μg, but not 0.1 μg, of DNR or DXRthere was a significant reduction in mean scotopic and photopic b-waveamplitudes. These data indicate that while DNR and DXR strongly suppressocular NV, bolus injections of free drugs can cause retinal toxicity.

Retinal Toxicity after Intraocular Injection of Digoxin

It has been previously demonstrated that intraocular injections of0.01-0.25 μg of digoxin suppress HIF-1 transcriptional activity andocular NV (Yoshida, T. et al. FASEB J. 24:1759-1767 (2010)). To explorewhether the deleterious effects of DXR and DNR on retinal function mightbe related to their suppression of HIF-1 activity, the effects ofintraocular injection of 0.25 and 0.05 μg of digoxin on retinal functionwere measured. One week after intraocular injection of 0.25 μg ofdigoxin, there was a significant reduction in mean scotopic a-waveamplitude, mean scotopic b-wave amplitude, and mean photopic b-waveamplitude. There was also a reduction in outer nuclear layer thicknessat 3 of 6 measurement locations in the retina, indicating death ofphotoreceptor cells. These results are consistent with substantialtoxicity after intraocular injection of 0.25 μg of digoxin. Intraocularinjection of 0.05 μg of digoxin was less toxic, but still causedsignificant reduction in mean scotopic and photopic b-wave amplitudes.Thus, for both anthracyclines and digoxin, injection of free drug intothe eye carries risk of retinal toxicity.

Effect of DXR Polymer Nanoparticles on Ocular NV

The effect of intraocular injection of DXR nanoparticles was firsttested in mice with laser-induced choroidal NV. After laser-inducedrupture of Bruch's membrane, C57BL/6 mice received an intraocularinjection of 10, 1, or 0.1 μg of DXR-PSA-PEG₃ nanoparticles. Fundusphotos of the animals that received 1 μg of DXR-PSA-PEG₃ nanoparticlesshowed a large orange mass of nanoparticles overlying the posteriorretina 1 day after injection that decreased slowly over time and wasstill readily visible on day 14. Particles remained visible over periodsof time as long as five weeks.

In mice perfused with fluorescein-labeled dextran to visualize choroidalNV by fluorescence microscopy at day 14, the area of choroidal NVappeared smaller in eyes given an intraocular injection of DXR-PSA-PEG₃nanoparticles compared to fellow eyes injected with PBS. Image analysisconfirmed that compared to eyes injected with PBS, the mean area ofchoroidal NV was significantly less in eyes injected with 10, 1, or 0.1μg of DXR-PSA-PEG₃ nanoparticles (FIG. 3A).

The effect of DXR-PSA-PEG₃ nanoparticles on already establishedchoroidal NV was investigated by allowing the NV to grow for 7 days andthen injecting 1 μg of DXR-PSA-PEG₃ nanoparticles. Seven days afterinjection, eyes injected with DXR-PSA-PEG₃ nanoparticles had a mean areaof choroidal NV that was significantly less than that seen in controleyes injected with PBS, and also significantly less than the baselineamount of choroidal NV that was present at 7 days (FIG. 3B). Thisindicates that DXR-PSA-PEG₃ nanoparticles cause regression ofestablished choroidal NV.

The DXR-PSA-PEG₃ nanoparticle formulation was also investigated using amodel of ischemia-induced retinal neovascularization (Smith, L. E. H. etal. Invest. Ophthalmol. Vis. Sci. 35:101-111 (1994)). Intraocularinjections of 1 μg of DXR-PSA-PEG₃ nanoparticles significantly reducedthe mean area of retinal NV compared to fellow eyes injected with PBS(FIG. 4).

Prolonged Suppression of NV after Intraocular Injection of DXR PolymerNanoparticles in Rho/VEGF Transgenic Mice

Rho/VEGF transgenic mice, in which the rhodopsin promoter drivesexpression of VEGF in photoreceptors, have sustained expression of VEGFstarting at postnatal day (P) 7, and provide an excellent model to testthe duration of activity of a therapeutic agent (Okamoto, N. et. al. Am.J. Pathol. 151:281-291 (1997)).

At P14, hemizygous rho/VEGF mice were given an intraocular injection of10 μg of DXR-PSA-PEG₃ nanoparticles in one eye and PBS in the felloweye. At 4 (FIG. 5A) or 5 weeks (FIG. 5B), the mean area of subretinal NVwas significantly less in DXR nanoparticle-injected eyes thanvehicle-injected fellow eyes.

Intraocular Injection of 1 or 10 μg of DXR Nanoparticles Did not CauseToxicity as Measured by ERG or ONL Thickness

At 14 days after intraocular injection of 10 μg of DXR-PSA-PEG₃, therewas no significant difference in scotopic or photopic b-wave amplitudescompared to PBS-injected eyes. There was also no difference in outernuclear layer thickness indicating that DXR nanoparticles did not causephotoreceptor cell death.

Example 3 Pharmacokinetic Study in Rabbits

Materials and Methods

Preparation of the PEG₃-PSA Polymer

(Polyethylene glycol) 3-co-poly(sebacic acid) (PEG3-PSA) was synthesizedby melt condensation. Briefly, sebacic acid was refluxed in aceticanhydride to form sebacic acid prepolymer (Acyl-SA). Polyethylene glycol(PEG₃) was prepared by mixing CH₃O-PEG-NH2 (2.0 g), citric acid (26 g),dicyclohexylcarbodiimide (DCC; 83 mg) and 4-(dimethylamino)pyridine(DMAP, 4.0 mg) which were added to 10 mL methylene chloride, stirredovernight at room temperature, then precipitated and washed with ether,and dried under vacuum. Next, acyl-SA (90% w/w) and PEG₃ (10% w/w) werepolymerized at 180° C. for 30 mins. Nitrogen gas was swept into theflask for 30 seconds every 15 minutes. Polymers were cooled to ambienttemperature, dissolved in chloroform and precipitated into excesspetroleum ether. The precipitate was collected by filtration and driedunder vacuum to constant weight, to produce the PEG3-PSA polymer.

Preparation of the DXR-PSA-PEG₃ Nanoparticles and Microparticles

To prepare DXR-PSA-PEG₃ nanoparticles, 80 mg PEG₃-PSA was dissolved in 6mL dichloromethane (DCM) and 20 mg doxorubicin hydrochloride (DXR)(NetQem LLC, Durham, N.C.) was dissolved in 2 mL dimethylsulfoxide(DMSO). The solutions of polymer and drug were mixed and kept at 50° C.for 30 min. The resulting mixture was homogenized in 50 mL of 1%polyvinyl alcohol (PVA) solution (25 kDa, Polyscience, Niles, Ill.) at10,000 rpm for 3 min using a L4RT homogenizer (Silverson Machines, EastLongmeadow, Mass.). The particle suspension was stirred at roomtemperature for 2 hours to remove dichloromethane. The particles werecollected by centrifugation (20,000×g for 20 minutes at 4° C.) andwashed thrice with ultrapure water prior to lyophilization.

DXR-PSA-PEG₃ microparticles were prepared in a similar fashion. Briefly,200 mg PEG₃-PSA was dissolved in 3 mL DCM and mixed with 40 mg DXRdissolved in 1.5 mL DMSO. Following incubation at 50° C. for 30 min, themixture was homogenized in 100 mL of PVA at 3,000 rpm for 1 min. Afterstirring for 2 hr, particles were collected by centrifugation (9,000×gfor 25 minutes) and washed thrice before lyophilization.

Particle Characterization

Particle size was determined using a Coulter Multisizer IV(Beckman-Coulter Inc., Fullerton, Calif.). Greater than 100,000particles were sized for each batch of microparticles to determine themean particle diameter. Particle morphology was characterized byscanning electron microscopy (SEM) using a cold cathode field emissionSEM (JEOL JSM-6700F, Peabody, Mass.). Drug loading was determined bydissolving dry powder of the particles in DCM and DMSO and theabsorbance was measured using a UV spectrophotometer at 490 nm.

Animal Procedures

Pigmented, Dutch-Belted rabbits were used for these studies (n=10).Animals were treated in accordance with the Association for Research inVision and Ophthalmology Statement of Use of Animals in Ophthalmic andResearch and the guidelines of the Johns Hopkins University Animal Careand Use Committee. For intraocular injections and collection of aqueoushumor, animals were anesthetized with an intramuscular injection ofketamine (25 mg/kg) and xylazine (2.5 mg/Kg). When sedated, the pupilswere dilated with 2.5% phenylephrine hydrochloride and 10% tropicamide.Ocular surface anesthesia was performed using topical instillation of0.5% proparacaine hydrochloride.

For the injections, a 26-gauge needle was carefully introduced into thevitreous cavity, 1.5 mm posterior to the superotemporal limbus, with theneedle tip directed into the mid-vitreous. A volume of 0.1 mL ofGB-AMD-101 micro or nanoparticle suspension was delivered to the righteyes, and 0.1 mL vehicle (PBS) was delivered to the left eyes. Theneedle was held in place for 10 seconds before withdrawal to preventreflux from the entry site. Animals were returned to their cages andmonitored until anesthesia was reversed. At the indicated times, aqueoushumor was withdrawn (˜0.1 mL) by inserting a 30-gauge needle through thelimbus and removing the aqueous humor. The samples were stored at −80°C. until use. At the end of the study (Day 105 for thenanoparticle-treated animals and Day 115 for the microparticle-treatedanimals), animals were euthanized using a pentobarbital-based euthanasia(>150 mg/Kg). Animals were enucleated and vitreous was isolated andstored at −80° C. until use.

HPLC Quantitation of Released Drug Conjugate in Rabbit Aqueous Humor andVitreous Samples

Prior to quantitation of the drug content by HPLC, 100 μL of aqueoushumor sample or vitreous sample was mixed with 200 μL, of methanol andincubated at 4° C. for 3 hr. After centrifugation (15,000×g, 10 min) andfiltration through a 0.2 μm PTFE filter, 150 μL of the filtrate wasinjected into a Waters HPLC system equipped with a c18 reverse phasecolumn (5 μm, 4.6×250 mm; Grace, Deerfield Ill.). Released drugconjugate was eluted by an isocratic mobile phase containing water andacetonitrile (60%:40%, v/v) at 1 mL/min and detected using afluorescence detector (excitation wavelength: 500 nm, emissionwavelength: 551 nm). The estimated limit of detection was 10 ng/mL. Aseries of DXR aqueous solutions at different concentrations were used ascalibration standards. The data was analyzed using Empower 3chromatography data software (Waters Corporation, Milford Mass.).

Results

DXR-PSA-PEG₃ Microparticles and Nanoparticles

Microparticles and nanoparticles composed of the DXR-SA-PEG₃ conjugatewere synthesized and characterized as described. Particles were sizedprior to lyophilization and following reconstitution in vehicle (PBS).The microparticles displayed a mean size of 27.2+1.0 um, and thenanoparticles, 0.98+0.02 um prior to lyophilization (Table 1; FIG. 6).The average drug loading of the microparticles was 13% and thenanoparticles was 20% (Table 1).

TABLE 1 Characterization of DXR-PSA-PEG₃ Micro and NanoparticlesParticle Diameter by Volume Average Sample ID Post- Drug Prior tolyophi- reconstitution Loading Type lization in PBS (w/w) Microparticles27.2 ± 10.4 μm 24.3 ± 8.3 μm 13% Nanoparticles 0.98 ± 0.74 μm  3.7 ± 2.0μm 20%

SEM analyses demonstrated discrete particles of the expected size. FIGS.6A and 6B show the size distribution by volume of the microparticles andnanoparticles, respectively.

Duration of Drug Release Following IVT Administration to Rabbits

Rabbits received an intravitreal injection (0.1 mL) of the DXR-PSA-PEG₃microparticles or nanoparticles into their right eyes and vehicle alone(PBS) into their left eyes. At the indicated times, aqueous humor wascollected (˜0.1 mL) and analyzed for the presence of released drugconjugate using a quantitative HPLC-based assay. On Day 115(microparticle group) or Day 105 (nanoparticle group), animals wereeuthanized and aqueous humor and vitreous was collected. The releaseddrug levels in the AH were compared to that in the vitreous for eachanimal.

All rabbits displayed sustained drug release following intravitrealparticle administration (FIG. 7A, Table 2).

TABLE 2 Pharmacokinetics of Intravitreal Delivery of DXR-PSA-PEG₃Particles to Rabbits. DXR-PSA-PEG₃ Microparticles DXR-PSA-PEG₃Nanoparticles DXR Conc. in Aqueous DXR Conc. in Aqueous Day Humor DayHumor 1 4.74 ± 2.23 μg/mL 1 6.91 ± 2.40 μg/mL 7 3.45 ± 1.76 μg/mL 8 2.51± 1.11 μg/mL 17 1.63 ± 0.65 μg/mL 18 1.51 ± 0.77 μg/mL 31 0.78 ± 0.52μg/mL 33 0.75 ± 0.41 μg/mL 64 0.16 ± 0.21 μg/mL 57 0.37 ± 0.27 μg/mL 920.21 ± 0.45 μg/mL 97 0.22 ± 0.26 μg/mL 115 00.5 ± 0.08 μg/mL 105 0.13 ±0.18 μg/mL Data presented as mean ± SD.

Levels well above the limit of quantitation of the HPLC assay (10 ng/mLor 20 nM) were observed in the AH of both the microparticle andnanoparticle-treated animals for the duration of the study, 115 and 105days, respectively (FIG. 7A). Direct comparison of the released druglevels in the AH compared to the vitreous revealed that vitreous levelswere significantly higher than those measured in the AH, up to 188 timeshigher in the vitreous compared to the AH (Table 3, FIG. 7B). The meanreleased drug levels for the microparticle-treated animals at Day 115were 0.09+0.13 uM in the AH and 7.12+12.92 uM in the vitreous. For thenanoparticle-treated animals at Day 105 mean released drug levels were0.23+0.31 uM in the AH and 11.91+10.45 uM in the vitreous (Table 3).Drug levels in the vitreous were 77-90 times higher than drug levelsmeasured in the AH.

FIG. 7A is a graph showing the amount of released DXR drug conjugate(nM) as a function of time (days) in the aqueous humor (AH) of rabbitstreated with microparticles and nanoparticles injected into thevitreous. FIG. 7B is a bar graph comparing the released drug amounts inthe aqueous humor (AH) and vitreous of nanoparticle andmicroparticle-treated rabbits at days 105 and 115, respectively.

TABLE 3 Comparison of Released Drug Levels in the Aqueous Humor vsVitreous DXR concentration Ratio (uM) Aqueous Humor Vitreous Vitreous/Treatment ug/mL uM ug/mL uM Aqueous Microparticles Rabbit 1 0.020 0.0340.34 0.59 17 Rabbit 2 0.184 0.317 17.50 30.17 95 Rabbit 3 0.014 0.0241.75 3.01 125 Rabbit 4 0.030 0.052 0.79 1.37 27 Rabbit 5 0.002 0.0040.27 0.47 122 Mean 0.05 ± 0.09 ± 4.13 ± 7.12 ± 77 ± 0.08 0.13 7.50 12.9252 Nanoparticles Rabbit 1 0.10 0.17 4.81 8.30 50 Rabbit 2 0.06 0.10 1.652.84 29 Rabbit 3 0.45 0.77 17.32 29.86 39 Rabbit 4 0.03 0.06 6.31 10.88188 Rabbit 5 0.03 0.05 4.44 7.66 144 Mean 0.13 ± 0.23 ± 6.91 ± 11.91 ±90 ± 0.18 0.31 6.06 10.45 72 Data presented as mean ± SD.

Intravitreal delivery of DXR-PSA-PEG₃ micro or nanoparticles to rabbiteyes resulted in long-term drug release, sustained for at least 115 or105 days, respectively, the duration of the study. The released druglevels measured in the vitreous where much higher than those measured inthe aqueous humor, an average of 77-90-fold higher.

These data demonstrate sustained release from of DXR-PSA-PEG₃ whendelivered intraocularly and suggest that of DXR-PSA-PEG₃ will be apromising therapy for the treatment of NV ocular diseases including NVAMD.

Example 4 Synthesis and In Vitro Evaluation of Fully Biodegradable ofDXR-PSA-PEG₃ Rods

Rod-shaped of DXR-PSA-PEG₃ were successfully produced with a diameter of0.5 mm, a length of 0.5 cm, and a mass of 1 mg, with three doxorubicin(DXR) drug loading levels, 10%, 30%, and 50%. DXR conjugate release invitro demonstrated release sustained for at least 25 days with all threerod types.

Materials and Methods

Preparation of PEG₃-PSA Polymer

(Polyethylene glycol)₃-co-poly(sebacic acid) (PEG₃-PSA) was synthesizedby melt condensation. Briefly, sebacic acid was refluxed in aceticanhydride to form sebacic acid prepolymer (Acyl-SA). Polyethylene glycol(PEG₃) was prepared by mixing CH₃O-PEG-NH₂ (2.0 g), citric acid (26 g),dicyclohexylcarbodiimide (DCC; 83 mg) and 4-(dimethylamino)pyridine(DMAP, 4.0 mg) which were added to 10 mL methylene chloride, stirredovernight at room temperature, then precipitated and washed with ether,and dried under vacuum. Next, acyl-SA (90% w/v) and PEG₃ (10% w/v) werepolymerized at 180° C. for 30 minutes. Nitrogen gas was swept into theflask every 30 seconds for 15 minutes. Polymers were cooled to ambienttemperature, dissolved in chloroform and precipitated into excesspetroleum ether. The precipitate was collected by filtration and driedunder vacuum to constant weight, to produce the PEG₃-PSA polymer.

Preparation of DXR-PSA-PEG₃ Rod

To prepare of DXR-PSA-PEG₃ rods, three different concentrations of DXRwere used to produce rods with drug loading levels of 10%, 30% and 50%(w/w). For the 10%, 30% and 50% drug loaded rods, PEG₃-PSA anddoxorubicin hydrochloride (DXR) (NetQem LLC, Durham, N.C.) were added toCHCl₃ at ratios of 9:1, 7:3, and 1:1 (w/w). The PEG₃-PSA and DXR wereincubated at 50° C. for one hour after which the CHCl₃ was removed byvacuum. The reaction product was grated to a fine powder and thencompressed into a glass tube, with a diameter of 0.5 mm, which was usedas a mold. The rods were extruded from the mold and cut to 0.5 cmlengths. Each rod weighed approximately 1 mg (0.9-1.2 mg).

In Vitro Drug Release

One rod (˜1 mg) was added to 1 ml of phosphate buffered saline (PBS, pH7.4) and incubated at 37° C. on a rotating platform (140 RPM). Atselected time points, supernatant was collected and fresh PBS added.DXR-conjugate concentration was measured by absorbance at 480 nm.

Results

Rod-shaped of DXR-PSA-PEG₃, were produced with three different drugloading levels, 10%, 30%, and 50%. The DXR-PSA-PEG₃ conjugates wereformed into rods with a diameter of 0.5 mm, a length of 0.5 cm, and amass of 1 mg.

The duration of in vitro drug release was evaluated using the ofDXR-PSA-PEG₃ rods, with drug loading levels of 10%, 30%, and 50% (FIG.8). Drug release from all three rods was sustained for at least 25 days.

These data demonstrate that the synthesis of rod-shaped DXR-PSA-PEG₃conjugates is possible. Rods composed of DXR-PSA-PEG₃ conjugates withdifferent drug concentrations were successfully synthesized, and allrods displayed sustained drug release in vitro. These data also suggestthat rods of differing sizes, mass, and drug content can be produced anddrug release rates optimized to obtain the most efficacious drugdelivery profile for each delivered drug and for each therapeuticindication.

Example 5 Production of DXR-PCPH-PSA-PEG₃ Polymer Conjugates

Microparticles composed of a fully biodegradable DXR-PSA-PCPH-PEG₃polymer drug conjugate were synthesized and displayed a slower drugrelease rate and more sustained drug release duration compared to theDXR-PSA-PEG₃ microparticles. The addition of CPH to the polymerincreased the hydrophobicity of polymer-drug conjugate which resulted ina prolonged the duration of drug release, presumably due to a reductionin DXR solubility.

Materials and Methods

Synthesis of 1,6-bis(p-carboxyphenoxy)hexane (CPH)

1,6-bis(p-carboxyphenoxy)hexane (CPH) was synthesized as described byConix (1966). Briefly, p-hydroxybenzoic acid (27.6 g) and sodiumhydroxide (16.0 g) in water (80 mL) were stirred and heated to refluxtemperature. 1,6-dibromohexane (96%, 15.7 mL) was added over a period of30 min while maintaining at reflux temperature and refluxed for anadditional 3.5 hours. Sodium hydroxide (4.0 g) dissolved in water (10mL) was added to the mixture and refluxed for another 2 hours beforeallowing the reaction mixture to stand overnight at room temperature.The disodium salt was isolated by filtration, washed with 40 mL ofmethanol, and dissolved in distilled water. The solution was warmed to60-70° C. and acidified with 6 N sulfuric acid. The dibasic acid wasisolated by filtration and dried to constant weight under vacuum.

Synthesis of PreCPH

1,6-bis(p-carboxyphenoxy)hexane (CPH) (10.0 g) was refluxed in 200 mL ofacetic anhydride for 30 min under nitrogen, followed by removal ofunreacted diacid by filtration and solvent by evaporation. The residuewas recrystallized from dimethylformamide and ethyl ether, washed withdry ethyl ether, and dried to constant weight under vacuum.

Synthesis of PEG₃-PSA-PCPH Prepolymer

(Polyethylene glycol)₃-co-poly(sebacic acid) co-poly(CPH) (PEG₃-SA-CPH)was synthesized by melt condensation. Briefly, sebacic acid was refluxedin acetic anhydride to form sebacic acid prepolymer (Acyl-SA).Polyethylene glycol (PEG₃) was prepared by mixing CH₃O-PEG-NH₂ (2.0 g),citric acid (26 g), dicyclohexylcarbodiimide (DCC; 83 mg) and4-(dimethylamino)pyridine (DMAP, 4.0 mg) which were added to 10 mLmethylene chloride, stirred overnight at room temperature, thenprecipitated and washed with ether, and dried under vacuum. Next, PEG₃(10% w/v), acyl-SA (60% w/v), and preCPH (30% w/v), were polymerized at180° C. for 30 minutes. Nitrogen gas was swept into the flask every 30second for 15 minutes. Polymers were cooled to ambient temperature,dissolved in chloroform and precipitated into excess petroleum ether.The precipitate was collected by filtration and dried under vacuum toconstant weight, to produce the PEG₃-PSA-PCPH prepolymer.

Preparation of DXR-PSA-PCPH-PEG₃ Microparticles

To prepare DXR-PSA-PCPH-PEG₃ microparticles, 200 mg PEG₃-PSA-PCPH wasdissolved in 3 mL dichloromethane (DCM) and mixed with 40 mg doxorubicinhydrochloride (DXR) (NetQem LLC, Durham, N.C.) dissolved in 1.5 mL DMSO.Following incubation at 50° C. for 30 min, the mixture was homogenizedin 100 mL of PVA at 3,000 rpm for 1 min. After stirring for 2 hr,particles were collected by centrifugation (9,000×g for 25 minutes) andwashed thrice before lyophilization.

Particle Characterization

Particle size was determined using a Coulter Multisizer IV(Beckman-Coulter Inc., Fullerton, Calif.). Greater than 100,000particles were sized for each batch of microparticles to determine themean particle diameter.

In Vitro Drug Release

DXR-PSA-PCPH-PEG₃ microparticles (2 mg) were suspended in phosphatebuffered saline (PBS, pH 7.4), and incubated at 37° C. on a rotatingplatform (140 RPM). At selected time points, supernatant was collectedby centrifugation (13,500×g for 5 min) and particles were resuspended infresh PBS. DXR-conjugate was measured by absorbance at 480 nm.

Results

DXR-PSA-PCPH-PEG₃ microparticles were synthesized and displayed a meansize of 24.3±8.7 um with a drug loading level of 13.9%.

The duration of in vitro drug release was compared between theDXR-PSA-PCPH-PEG₃ microparticles and the DXR-PSA-PEG₃ microparticles(mean size 22.5±8.3 um). The DXR-PSA-PEG₃ microparticles showed drugrelease sustained for 45 days while the DXR-PSA-PCPH-PEG₃ microparticlesdemonstrated a slower drug release rate and drug release sustained forover 75 days (FIG. 9).

Microparticles composed of a fully biodegradable DXR-PSA-PCPH-PEG₃polymer drug conjugate were synthesized. The DXR-PSA-PCPH-PEG₃microparticles displayed a slower drug release rate and more sustaineddrug release duration compared to the DXR-PSA-PEG₃ microparticles,particles lacking the addition of the PCPH polymer. The addition of PCPHto the polymer increased the hydrophobicity of the released drugconjugate which is expected to reduce the solubility of DXR, andresulted in a prolonged duration of drug release.

These data demonstrate that by altering the polymer chemistry toincrease the hydrophobicity of the released drug conjugate, the leveland duration of drug release can be modified, suggesting that theseparameters can be optimized to obtain the most efficacious drug deliveryprofile for each delivered drug and for each therapeutic indication.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meanings as commonly understood by one of skill in the artto which the disclosed invention belongs. Publications cited herein andthe materials for which they are cited are specifically incorporated byreference.

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

We claim:
 1. A polymeric conjugate defined by one of the followingformulae(A-X)_(m)—Y—((Z)_(o)—(X)_(p)-(A)_(q))_(n) wherein A represents,independently for each occurrence, a HIF-1 inhibitor; X represents,independently for each occurrence, a hydrophobic polymer segment; Y isabsent, or represents a branch point; Z represents, independently foreach occurrence, a hydrophilic polymer segment; and o, p, and q areindependent 0 or 1; m is an integer between one and twenty; and n is aninteger between zero and twenty, with the proviso that A is notdoxorubicin when m and n are both equal to one, wherein solubility ofthe conjugate can be controlled by modifying the solubility of thepolymer portion and/or the branched point Y to minimize soluble HIF-1inhibitor concentration.
 2. The polymeric conjugate of claim 1, whereinA is an anthracycline.
 3. The polymeric conjugate of claim 1, wherein Zis selected from the group consisting of a poly(alkylene glycol), apolysaccharide, poly(vinyl alcohol), polypyrrolidone, a polyoxyethyleneblock copolymer (PLURONIC®), and copolymers thereof.
 4. The polymericconjugate of claim 3, wherein Z for each occurrence comprisespolyethylene glycol.
 5. The polymeric conjugate claim 1, wherein X isbiodegradable.
 6. The polymeric conjugate of claim 5, wherein X isselected from the group consisting of polyesters, polycaprolactone,polyanhydrides, and copolymers thereof.
 7. The polymeric conjugate ofclaim 6, wherein X comprises a polyanhydride.
 8. The polymeric conjugateof claim 7, wherein X comprises polysebacic anhydride.
 9. The polymericconjugate of claim 7, wherein X comprises 1,6bis(p-carboxyphenoxy)hexane (CPH) or a combination of poly-CPH(PCPH) andpolysebacic anhydride.
 10. The polymeric conjugate of claim 1, wherein Yis one of the following:


11. The polymeric conjugate of claim 10, wherein Y is citric acid. 12.The polymeric conjugate of claim 1, defined by the following formulaA-X—YZ)_(n) wherein A represents, independently for each occurrence, aHIF-1 inhibitor; X represents, a hydrophobic polymer segment; Yrepresents a branch point; Z represents, independently for eachoccurrence, a hydrophilic polymer segment; and n is an integer betweenone and ten or two and ten.
 13. The polymeric conjugate of claim 12,wherein A is an anthracycline.
 14. The polymeric conjugate of claim 12,wherein Z is selected from the group consisting of a poly(alkyleneglycol), a polysaccharide, poly(vinyl alcohol), polypyrrolidone, apolyoxyethylene block copolymer, and copolymers thereof.
 15. Thepolymeric conjugate of claim 14, wherein Z for each occurrence comprisespolyethylene glycol.
 16. The polymeric conjugate claim 12, wherein X isbiodegradable.
 17. The polymeric conjugate of claim 16, wherein X isselected from the group consisting of polyesters, polycaprolactone,polyanhydrides, and copolymers thereof.
 18. The polymeric conjugate ofclaim 17, wherein X comprises a polyanhydride.
 19. The polymericconjugate of claim 18, wherein X comprises polysebacic anhydride. 20.The polymeric conjugate of claim 17, wherein X comprises 1,6bis(p-carboxyphenoxy)hexane (CPH) or a combination of CPH andpolysebacic anhydride
 21. The polymeric conjugate of claim 12, wherein nis between 2 and
 6. 22. The polymeric conjugate of claim 21, wherein nis
 3. 23. The polymeric conjugate of claim 12, wherein Y is one of thefollowing:


24. The polymeric conjugate of claim 23, wherein Y is citric acid. 25.The polymeric conjugate of claim 1, wherein the polymeric conjugate isdefined by Formula I

wherein A is a HIF-1 inhibitor; L represents, independently for eachoccurrence, an ether (e.g., —O—), thioether (e.g., —S—), secondary amine(e.g., —NH—), tertiary amine (e.g., —NR—), secondary amide (e.g.,—NHCO—; —CONH—), tertiary amide (e.g., —NRCO—; —CONR—), secondarycarbamate (e.g., —OCONH—; —NHCOO—), tertiary carbamate (e.g., —OCONR—;—NRCOO—), urea (e.g., —NHCONH—; —NRCONH—; —NHCONR—⁻, —NRCONR—), sulfinylgroup (e.g., —SO—), or sulfonyl group (e.g., —SOO—); R is, individuallyfor each occurrence, an alkyl, cycloalkyl, heterocycloalkyl, alkylaryl,alkenyl, alkynyl, aryl, or heteroaryl group, optionally substituted withbetween one and five substituents individually selected from alkyl,cyclopropyl, cyclobutyl ether, amine, halogen, hydroxyl, ether, nitrile,CF₃, ester, amide, urea, carbamate, thioether, carboxylic acid, andaryl; PEG represents a polyethylene glycol chain; and X represents ahydrophobic polymer segment.
 26. The polymeric conjugate of claim 25,wherein X is biodegradable.
 27. The polymeric conjugate of claim 25,wherein X is selected from the group consisting of polyesters,polycaprolactone, polyanhydrides, and copolymers thereof.
 28. Thepolymeric conjugate of claim 27, wherein X comprises a polyanhydride.29. The polymeric conjugate of claim 28, wherein X comprises polysebacicanhydride.
 30. The polymeric conjugate of claim 26, wherein X comprises1,6 bis(p-carboxyphenoxy)hexane (CPH) or a combination of CPH andpolysebacic anhydride.
 31. The polymeric conjugate of claim 25, whereinone or more of L are amides or esters.
 32. The polymeric conjugate ofclaim 31, wherein A in an anthracycline.
 33. The polymeric conjugate ofclaim 1, wherein the polymeric conjugate is defined by Formula IA

wherein A is a HIF-1 inhibitor; D represents, independently for eachoccurrence, O or NH; PEG represents a polyethylene glycol chain; and Xis represents a hydrophobic polymer segment.
 34. The polymeric conjugateof claim 33, wherein A in an anthracycline.
 35. The polymeric conjugateof claim 33, wherein X is biodegradable.
 36. The polymeric conjugate ofclaim 35, wherein X comprises a polyanhydride.
 37. The polymericconjugate of claim 36, wherein X comprises polysebacic anhydride. 38.The polymeric conjugate of claim 36, wherein X comprises 1,6bis(p-carboxyphenoxy)hexane (CPH) or a combination of CPH andpolysebacic anhydride
 39. A population of micro- and/or nanoparticlescomprising the conjugate of claim
 1. 40. A population of micro- and/ornanoparticles comprising the conjugate of claim
 12. 41. A population ofmicro- and/or nanoparticles comprising the conjugate of claim
 25. 42. Aformulation comprising the polymeric conjugate of claim 1, or particlesthereof in a pharmaceutically acceptable carrier.
 43. A formulationcomprising the polymeric conjugate of claim 12, or particles thereof.44. A formulation comprising the polymeric conjugate of claim 25, or theparticles of claim
 43. 45. The formulation of claim 42, comprising aconjugate defined by the following formulaA-X wherein A is a HIF-1 inhibitor; and X is a hydrophobic polymersegment.
 46. The formulation of claim 45, wherein A in an anthracycline.47. The formulation of claim 45, wherein X comprises a polyanhydride.48. The formulation of claim 57, wherein X comprises polysebacicanhydride.
 49. The polymeric conjugate of claim 47, wherein X comprises1,6 bis(p-carboxyphenoxy)hexane (CPH) or a combination of CPH andpolysebacic anhydride.
 50. A method of treating or preventing a diseaseor disorder involving aberrant vascularization, comprising administeringto a patient in need thereof a formulation of claim 42, in apharmaceutically acceptable excipient, as nano or microparticles or inan implant or depo.
 51. A method of treating a disease or disorder ofthe eye comprising administering to the eye of a patient in need thereofthe formulation of claim 42, in an excipient, as nano or microparticlesor in an implant or depo pharmaceutically acceptable for administrationto the eye.
 52. The method of claim 51, wherein the disease or disorderof the eye is an intraocular neovascular disease, disorder, or injury.53. The method of claim 52, wherein the intraocular neovascular diseaseor disorder is selected from the group consisting of age-related maculardegeneration associated with choroidal neovascularization, proliferativediabetic retinopathy, proliferative vitreoretinopathy, retinopathy ofprematurity, pathological myopia, von Hippel-Lindau disease, presumedocular histoplasmosis syndrome (POHS), and conditions associated withischemia such as branch retinal vein occlusion, central retinal veinocclusion, branch retinal artery occlusion, and central retinal arteryocclusion, neovascularization associated with a tumor,neovascularization associated with an ocular wound, retinalneovascularization, corneal graft rejection, complications from surgerythat cause neovascularization, complications from injury that causeneovascularization, and combinations thereof.
 54. The method of claim53, wherein the disease or disorder is wet age-related maculardegeneration.
 55. The method of claim 53, wherein the disease ordisorder involves choroidal neovascularization.
 56. The method of claim55, wherein composition provides an effective amount of one or moreactive agents to decrease the area of choroidal neovascularization, asmeasured by fluorescein angiography, by at least 15%.
 57. The method ofclaim 53, wherein the disease or disorder involves retinalneovascularization.
 58. The method of claim 57, wherein compositionprovides an effective amount of one or more active agents to decreasethe area of retinal neovascularization, as measured by fluoresceinangiography, by at least 15%.